Pectate lyase variants

ABSTRACT

The present invention relates to pectate lyase variants exhibiting alterations relative to a parent enzyme exhibiting pectate lyase activity as its major enzymatic activity; to a method of producing such enzymes; and to methods for using such enzymes in the textile, detergent and cellulose fiber processing industries. Compared to the parent enzyme, the pectate lyase variants of the present invention exhibit improved stability in detergents.

TECHNICAL FIELD

The present invention relates to pectate lyase variants exhibitingalterations relative to a parent enzyme exhibiting pectate lyaseactivity as its major enzymatic activity; to a method of producing suchenzymes; and to methods for using such enzymes in the textile, detergentand cellulose fiber processing industries. Compared to the parentenzyme, the pectate lyase variants of the present invention may exhibitimproved stability in detergents.

BACKGROUND OF THE INVENTION

Pectin polymers are important constituents of plant cell walls. Pectinis a hetero-polysaccharide with a backbone composed of alternatinghomogalacturonan (smooth regions) and rhamnogalacturonan (hairyregions). The smooth regions are linear polymers of 1,4-linkedalpha-D-galacturonic acid. The galacturonic acid residues can bemethyl-esterified on the carboxyl group to a varying degree, usually ina non-random fashion with blocks of polygalacturonic acid beingcompletely methyl-esterified.

Pectolytic enzymes (pectinases) can be classified according to theirpreferential substrate, highly methyl-esterified pectin or lowmethyl-esterified pectin and polygalacturonic acid (pectate), and theirreaction mechanism, beta-elimination or hydrolysis. Pectinases can bemainly endo-acting, cutting the polymer at random sites within the chainto give a mixture of oligomers, or they may be exo-acting, attackingfrom one end of the polymer and producing monomers or dimers. Severalpectinase activities acting on the smooth regions of pectin are includedin the classification of enzymes provided by the Enzyme Nomenclature(1992) such as pectate lyase (EC 4.2.2.2), pectin lyase (EC 4.2.2.10),polygalacturonase (EC 3.2.1.15), exo-polygalacturonase (EC 3.2.1.67),exo-polygalacturonate lyase (EC 4.2.2.9) andexo-poly-alpha-galacturonosidase (EC 3.2.1.82).

Pectate lyases have been cloned from different bacterial genera such asErwinia, Pseudomonas, Klebsiella and Xanthomonas. Also from Bacillussubtilis (Nasser et al. (1993) FEBS 335:319-326) and Bacillus sp. YA-14(Kim et al. (1994) Biosci. Biotech. Biochem. 58:947-949) cloning of apectate lyase has been described.

The pectate lyases are generally characterised by an alkaline pH optimumand an absolute requirement for divalent cations, Ca²⁺ being the moststimulatory.

It is an object of the present invention to provide a cell-walldegrading enzyme variant, especially a pectate lyase enzyme variant,which exhibits improved performance over the parent pectate lyase whenapplied e.g. in detergents or in textile industry processes.

SUMMARY OF THE INVENTION

The inventors have now found that certain amino acid substitutions incell-wall degrading enzymes especially pectate lyases having a structureincluding a beta-helix result in enzyme variants having improvedperformance in the neutral or alkaline pH range compared to the parentenzyme. The pectate lyase variants of the invention, when used indetergent compositions, have improved storage stability i.e. lowersensitivity to detergents.

Thus, in a first aspect the present invention relates to a pectate lyasevariant comprising alterations at one or more positions selected fromthe group consisting of positions number: 5, 9, 11, 26, 28, 30, 31, 37,40, 45, 46, 47, 48, 49, 50, 51, 52, 54, 61, 64, 68, 69, 70, 71, 74, 75,76,79, 86, 87, 91, 99, 105, 106, 107, 111, 115, 116, 118, 122, 123, 134,136, 139, 140, 141, 146, 148, 156, 158, 170, 182, 185, 186, 189, 193,194, 196, 199, 201, 202, 204, 213, 215, 218, 224, 228, 229, 234, 235,237, 251, 256, 257, 258, 272, 277, 286, 295, 298, 301, 302, 303, 305,307, 308, 314, 316, 323, 324, 326, 331, 332, 333, 334, 335, 336, 337,338, 339, 340, 341, 349, 356, 357, 363, 366, 378, 381, 384, 386, 387,389, 390, 391, 393 and 397,

wherein the alteration(s) are independently

(i) an insertion of an amino acid downstream of the amino acid whichoccupies the position,

(ii) a deletion of the amino acid which occupies the position, or

(iii) a substitution of the amino acid which occupies the position witha different amino acid,

and wherein each position corresponds to a position of the amino acidsequence of the pectate lyase having the amino acid sequence of SEQ IDNO:2, and wherein the parent enzyme is the pectate lyase shown on SEQ IDNO:2 or a pectate lyase having at least 65% identity to the amino acidsequence of SEQ ID NO:2.

In a second aspect the present invention relates to a nucleic acidsequence encoding the pectate lyase variant.

In a third aspect of the invention there is provided an expressionvector.

In a fourth aspect of the present invention there is provided amicrobial host cell transformed with the abovementioned expressionvector.

In a fifth aspect of the present invention there is provided a methodfor improving the detergent stability of a pectate lyase, comprisingaltering one or more amino acids.

In a sixth aspect of the invention there are provided methods forproducing a pectate lyase variant according to the invention comprisingculturing a cell into which has been introduced an expression vector asdisclosed above, whereby said cell expresses the variant encoded by thenucleic acid sequence and recovering the pectate lyase variant.

The pectate lyase variant of the invention is useful for the treatmentof cellulosic material, especially cellulose-containing fiber, yarn,woven or non-woven fabric, treatment of mechanical paper-making pulps orrecycled waste paper, and for retting of fibres. The treatment can becarried out during the processing of cellulosic material into a materialready for garment manufacture or fabric manufacture, e.g. in thedesizing or scouring step; or during industrial or household launderingof such fabric or garment.

Accordingly, in further aspects the present invention relates to adetergent composition comprising a pectate lyase variant havingsubstantial cell-wall degrading activity; and to use of the pectatelyase variant of the invention for the treatment e.g. cleaning ofcellulose-containing fibers, yarn, woven or non-woven fabric.

Further, additional aspects of the invention relates to an enzymecomposition comprising the pectate lyase variant of the invention incombination with other enzymes, and to a cleaning or detergentcomposition, preferably a laundry or dish wash composition, comprisingthe pectate lyase variant of the invention.

The pectate lyase variant of the invention, is very effective for use inan enzymatic scouring process in the preparation of cellulosic materiale.g. for proper response in subsequent dyeing operations.

Another aspect of the invention relates to the processing of wine andjuice. The enzyme or enzyme preparation may be used in the treatment ofmash from fruits and vegetables in order to improve the extractabilityor degradability of the mash.

Further, an aspect of the invention is the application as an animal feedadditive. When added to feed containing plant material from soy bean,rape seed, lupin etc the pectate lyase variant significantly improvesthe in vivo break-down of plant cell wall material, whereby a betterutilization of the plant nutrients by the animal is achieved.

DEFINITIONS

Prior to discussing this invention in further detail, the followingterms and conventions will first be defined.

The term “wild-type enzyme” denotes an enzyme, which is endogenous to anaturally occurring microorganism such as a fungus or a bacterium foundin Nature.

The term “parent enzyme” as used herein means an enzyme in whichmodifications are being made to produce the enzyme variants of theinvention. A parent enzyme may be an enzyme isolated from a naturalsource, or an enzyme wherein previous modification(s) have been madewhile retaining the characteristic activity of the enzyme in question.The parent pectate lyase of the invention may be a wild-type pectatelyase.

The term “enzyme variant” means an enzyme comprising differences in itsamino acid sequence from that of the parent enzyme. The differencescomprise substitutions, deletions and/or insertions as compared to theparent enzyme.

The term “ortholog” (or “species homolog”) denotes a polypeptide orprotein obtained from one species that has homology to an analogouspolypeptide or protein from a different species.

The term “paralog” denotes a polypeptide or protein obtained from agiven species that has homology to a distinct polypeptide or proteinfrom that same species.

The term “expression vector” denotes a DNA molecule, linear or circular,that comprises a segment encoding a polypeptide of interest operablylinked to additional segments that provide for its transcription. Suchadditional segments may include promoter and terminator sequences, andmay optionally include one or more origins of replication, one or moreselectable markers, an enhancer, a polyadenylation signal, and the like.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both. The expression vector of the invention maybe any expression vector that is conveniently subjected to recombinantDNA procedures, and the choice of vector will often depend on the hostcell into which the vector it is to be introduced. Thus, the vector maybe an autonomously replicating vector, i.e. a vector existing as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

The term “recombinant expressed” or “recombinantly expressed” usedherein in connection with expression of a polypeptide or protein isdefined according to the standard definition in the art. Recombinantexpression of a protein is generally performed by using an expressionvector as described immediately above.

The term “isolated”, when applied to a polynucleotide molecule, denotesthat the polynucleotide has been removed from its natural genetic milieuand is thus free of other extraneous or unwanted coding sequences, andis in a form suitable for use within genetically engineered proteinproduction systems. Such isolated molecules are those that are separatedfrom their natural environment and include cDNA and genomic clones.Isolated DNA molecules of the present invention are free of other geneswith which they are ordinarily associated, but may include naturallyoccurring 5′ and 3′ untranslated regions such as promoters andterminators. The identification of associated regions will be evident toone of ordinary skill in the art (see for example, Dynan and Tijan,Nature 316:774-78, 1985). The term “an isolated polynucleotide” mayalternatively be termed “a cloned polynucleotide”.

When applied to a protein/polypeptide, the term “isolated” indicatesthat the protein is found in a condition other than its nativeenvironment. In a preferred form, the isolated protein is substantiallyfree of other proteins, particularly other homologous proteins (i.e.“homologous impurities” (see below)). It is preferred to provide theprotein in a greater than 40% pure form, more preferably greater than60% pure form. Even more preferably it is preferred to provide theprotein in a highly purified form, i.e., greater than 80% pure, morepreferably greater than 95% pure, and even more preferably greater than99% pure, as determined by SDS-PAGE.

The term “isolated protein/polypeptide may alternatively be termed“purified protein/polypeptide”.

The term “homologous impurities” means any impurity, e.g. anotherpolypeptide than the polypeptide of the invention, which originate fromthe homologous cell where the polypeptide of the invention is originallyobtained from.

The term “obtained from” as used herein in connection with a specificmicrobial source, means that the polynucleotide and/or polypeptide isproduced by the specific source, or by a cell in which a gene from thesource have been inserted.

The term “operably linked”, when referring to DNA segments, denotes thatthe segments are arranged so that they function in concert for theirintended purposes, e.g. transcription initiates in the promoter andproceeds through the coding segment to the terminator.

The term “polynucleotide” denotes a single- or double-stranded polymerof deoxyribonucleotide or ribonucleotide bases read from the 5′ to the3′ end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules.

The term “complements of polynucleotide molecules” denotespolynucleotide molecules having a complementary base sequence andreverse orientation as compared to a reference sequence. For example,the sequence 5′ ATGCACGGG 3′ is complementary to 5′ CCCGTGCAT 3′.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “promoter” denotes a portion of a gene containing DNA sequencesthat provide for the binding of RNA polymerase and initiation oftranscription. Promoter sequences are commonly, but not always, found inthe 5′ non-coding regions of genes.

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger peptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

The term “pectin” denotes pectate, polygalacturonic acid, and pectinwhich may be esterified to a higher or lower degree.

The term “pectinase” denotes a pectinase enzyme defined according to theart where pectinases are a group of enzymes that cleave glycosidiclinkages of pectic substances mainly poly(1,4-alpha-D-galacturonide andits derivatives (see reference Sakai et al., Pectin, pectinase andprotopectinase: production, properties and applications, pp 213-294 in:Advances in Applied Microbiology vol: 39, 1993).

Preferably a pectinase of the invention is a pectinase enzyme whichcatalyzes the random cleavage of alpha-1,4-glycosidic linkages in pecticacid also called polygalacturonic acid by transelimination such as theenzyme class polygalacturonate lyase (EC 4.2.2.2) (PGL) also known aspoly(1,4-alpha-D-galacturonide) lyase also known as pectate lyase.

The term “thermostability” or “thermal stability” is intended to meanthe stability of the protein to thermal influence. All enzyme proteinsdestabilizes and eventually degrades with increasing temperature, eachenzyme protein having a certain temperature range wherein the protein isstable and retains its enzymatic activity. Increased thermostabilitymeans that the enzyme protein may retain its enzymatic activity and/orexhibit a higher relative activity at increased temperatures.

The term “detergent stability” or “storage stability” is intended tomean the stability of the protein in a formulation containing detergentse.g. anionic surfactants. Anionic surfactants are characterized by thecombination of an anionic group and a hydrophobic tail. When binding tothe protein, a positively charged residue like Lysine or Arginine, and ahydrophobic area are thus likely interaction points. Similarly thedynamic of particularly flexible regions is opening up for theaccessibility to amino acids normally buried in the internal of theprotein. These residues are typically hydrophobic and are thusattractive for the tail of the surfactant. A chemical interactionbetween enzyme and surfactant will with high certainty leave the enzymeinactive. Thus improved detergent- or storage stability means that at acertain detergent concentration and temperature, a greater enzymaticactivity will be retained after a certain period of time (greaterresidual activity).

Accordingly, thermostability and detergent stability are two independentcharacteristics of a protein or an enzyme.

The pectate lyase variants of the invention having improved detergentstability may exhibit at least 120% (preferably at least 140%, morepreferably at least 160%, even more preferably at least 180%, even morepreferably at least 200%, most preferably at least 250% and inparticular at least 300%) residual activity compared to the parentpectate lyase, when subjected to the analysis method described inExample 1.

Protein Numbering

In the context of this invention, a specific numbering of amino acidresidue positions in cell-wall degrading enzymes, especially pectatelyase enzymes, are employed. For example, by aligning the amino acidsequences of known pectate lyases it is possible to unambiguously allotan amino acid position number to any amino acid residue in any pectatelyase enzyme, if its amino acid sequence is known.

Using the numbering system originating from the amino acid sequence ofthe pectate lyase encoded by the polynucleotide present in the plasmidof the strain Bacillus subtillis DSM 14218, disclosed in SEQ ID NO: 2,aligned with the amino acid sequence of a number of other pectatelyases, it is possible to indicate the position of an amino acid residuein a pectate lyase enzyme unambiguously.

In describing the various cell-wall degrading enzyme variants producedor contemplated according to this invention, the following nomenclaturesare adapted for ease of reference:

Substitutions

[Original amino acid; Position; Substituted amino acid] Accordingly, thesubstitution of serine with isoleucine in position 72 is designated asS72I.

Multiple mutations are separation by addition marks (“+”), e.g.M169I+F198V, representing mutations in positions 169 and 198substituting methionine (M) with isoleucine (I), and phenylalanine (F)with valine (V), respectively.

Deletions

A deletion of glycine in position 195 will be indicated by:

-   -   Gly195* or G195*

Correspondingly the deletion of more than one amino acid residue, suchas the deletion of glycine and leucine in positions 195 and 196 will bedesignated

-   -   Gly195*+Leu196* or G195*+L196*        Insertions

The insertion of an additional amino acid residue such as e.g. a lysineafter G195 is indicated by:

-   -   Gly195GlyLys or G195GK;

or, when more than one amino acid residue is inserted, such as e.g. aLys and Ala after G195 this will be indicated as:

-   -   Gly195GlyLysAla or G195GKA

In such cases the inserted amino acid residue(s) are numbered by theaddition of lower case letters to the position number of the amino acidresidue preceding the inserted amino acid residue(s). In the aboveexample the sequences 194 to 196 would thus be changed from:

-   -   194 195 196    -   A-G-L

to 194 195 195a 195b 196

-   -   A-G-K-A-L

In cases where an amino acid residue identical to the existing aminoacid residue is inserted it is clear that degeneracy in the nomenclaturearises. If for example a glycine is inserted after the glycine in theabove example this would be indicated by G195GG. The same actual changecould just as well be indicated as A194AG for the change from:

-   -   194 195 196    -   A-G-L

to

-   -   194 195 195a 196    -   A-G-G-L

or 194 194a 195 196

Such instances will be apparent to the skilled person and the indicationG195GG and corresponding indications for this type of insertions arethus meant to comprise such equivalent degenerate indications.

All positions referred to herein by pectate lyase numbering refer,unless otherwise stated, to the numbering described above, and aredetermined relative to the amino acid sequence of the pectate lyaseencoded by the polynucleotide present in the plasmid of the strainBacillus subtilis DSM 14218, disclosed in SEQ ID NO:2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is pertaining to variants of a parent pectatelyase (EC 4.2.2.2). The variants have improved properties compared tothe parent enzyme, especially the detergent stability or storagestability in detergent compositions is improved.

In the process of improving the properties of the parent pectate lyase,the inventors found that alterations of specific amino acids in theparent polypeptide backbone would significantly alter the detergentstability of the enzyme.

Polynucleotides

Within preferred embodiments of the invention it is contemplated that apolynucleotide encoding the parent enzyme of the pectate lyase variantof the invention will hybridize to similar sized regions of thecorresponding polynucleotide of SEQ ID NO:1, or a sequence complementarythereto, under at least medium stringency conditions, preferably highstringency conditions.

In particular polynucleotides of the invention will hybridize to adenatured double-stranded DNA probe comprising either the full variantsequence corresponding to positions 1-1200 of SEQ ID NO:1 with propersequence alterations corresponding to actual amino acid substitutionsmade or any probe comprising a variant subsequence thereof having alength of at least about 100 base pairs under at least medium stringencyconditions, but preferably at high stringency conditions as described indetail below. Suitable experimental conditions for determininghybridization at medium, or high stringency between a nucleotide probeand a homologous DNA or RNA sequence involves presoaking of the filtercontaining the DNA fragments or RNA to hybridize in 5× SSC (Sodiumchloride/Sodium citrate, Sambrook et al. 1989) for 10 min, andprehybridization of the filter in a solution of 5× SSC, 5× Denhardt'ssolution (Sambrook et al. 1989), 0.5% SDS and 100 microgram/ml ofdenatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed byhybridization in the same solution containing a concentration of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)Anal. Biochem. 132:6-13), 32P-dCTP-labeled (specific activity higherthan 1×109 cpm/microgram) probe for 12 hours at ca. 45 degrees Celsius.The filter is then washed twice for 30 minutes in 2× SSC, 0.5% SDS atleast 60 degrees Celsius (medium stringency), still more preferably atleast 65 degrees Celsius (medium/high stringency), even more preferablyat least 70 degrees Celsius (high stringency), and even more preferablyat least 75 degrees Celsius (very high stringency).

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using an X-ray film.

As previously noted, the polynucleotides encoding the pectate lyasevariants of the present invention include DNA and RNA. Methods forisolating DNA and RNA are well known in the art. DNA and RNA encodinggenes of interest can be cloned in Gene Banks or DNA libraries by meansof methods known in the art.

Polynucleotides encoding polypeptides having pectate lyase activity ofthe invention are then identified and isolated by, for example,hybridization or PCR. Species homologues of the parent pectate lyaseused in preparation of the pectate lyase variants of the invention canbe cloned using information and compositions provided by the presentinvention in combination with conventional cloning techniques. Forexample, DNA can be cloned using chromosomal DNA obtained from a celltype that expresses the protein. Suitable sources of DNA can beidentified by probing Northern blots with probes designed from thesequences disclosed herein. A library is then prepared from chromosomalDNA of a positive cell line. A DNA encoding a polypeptide having pectatelyase activity of the invention can then be isolated by a variety ofmethods, such as by probing with a complete or partial DNA or with oneor more sets of degenerate probes based on the disclosed sequences. ADNA can also be cloned using the polymerase chain reaction, or PCR(Mullis, U.S. Pat. No. 4,683,202), using primers designed from thesequences disclosed herein. Within an additional method, the DNA librarycan be used to transform or transfect host cells, and expression of theDNA of interest can be detected with an antibody (mono-clonal orpolyclonal) raised against the pectate lyase cloned from B. subtilisstrain deposited as IFO 3134, or by an activity test relating to apolypeptide having pectate lyase activity. Similar techniques can alsobe applied to the isolation of genomic clones.

The polypeptide encoding part of the DNA sequence cloned a plasmidpresent in Bacillus subtilis DSM 14218 and/or an analogue DNA sequenceof the invention may be cloned from a strain of the bacterial speciesBacillus subtilis, preferably the strain deposited as IFO 3134,producing the enzyme with pectin degrading activity, or another orrelated organism as described herein.

Alternatively, the analogous sequence may be constructed on the basis ofthe DNA sequence obtainable from a plasmid present in Bacillus subtilisDSM 14218 e.g. be a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the pectate lyase encoded by the DNA sequence, but whichcorresponds to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence (i.e. a variantof the pectin degrading enzyme of the invention).

Polypeptides

The sequence of amino acids no. 1-399 of SEQ ID No 2 is a mature pectatelyase sequence corresponding to a wild-type pectate lyase from thespecies Bacillus subtilis deposited as IFO 3134 (Institute forFermentation, Osaka, 17-85, Jusohonmachi 2-chome, Yodagawa-ku, Osaka532-8686, Japan).

The present invention also provides pectate lyase variants ofpolypeptides that are substantially homologous to the polypeptides ofSEQ ID NO:2 and its species homologs (paralogs or orthologs). The term“substantially homologous” is used herein to denote polypeptides having70%, more preferably at least 85%, and even more preferably at least90%, sequence identity to the sequence shown in SEQ ID NO:2 or itsorthologs or paralogs. Such polypeptides will more preferably be atleast 95% identical, and most preferably 98% or more identical to thesequence shown in SEQ ID NO:2 or its orthologs or paralogs. Percentsequence identity is determined by conventional methods, by means ofcomputer programs known in the art such as GAP provided in the GCGprogram package (Program Manual for the Wisconsin Package, Version 8,August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis.,USA 53711) as disclosed in Needleman, S. B. and Wunsch, C. D., (1970),Journal of Molecular Biology, 48, 443-453, which is hereby incorporatedby reference in its entirety. GAP is used with the following settingsfor polypeptide sequence comparison: GAP creation penalty of 3.0 and GAPextension penalty of 0.1.

Sequence identity of polynucleotide molecules is determined by similarmethods using GAP with the following settings for DNA sequencecomparison: GAP creation penalty of 5.0 and GAP extension penalty of0.3.

The parent pectate lyase is preferably derived from a microorganism,preferably from a bacterium, an archea or a fungus, especially from abacterium such as a bacterium belonging to Bacillus, preferably to analkalophilic Bacillus strain which may be selected from the groupconsisting of the species Bacillus subtilis and highly related Bacillusspecies in which all species preferably are at least 95%, even morepreferably at least 98%, homologous to Bacillus subtilis based onaligned 16S rDNA sequences. The parent pectate lyases of the inventionare substantially homologous to the polypeptides of SEQ ID NO:2 and itsspecies homologs (paralogs or orthologs). The term “substantiallyhomologous” is used herein to denote polypeptides having 70%, morepreferably at least 85%, and even more preferably at least 90%, sequenceidentity to the sequence shown in SEQ ID No:2 or its orthologs orparalogs. Such polypeptides will more preferably be at least 95%identical, and most preferably 98% or more identical to the sequenceshown in SEQ ID NO:2 or its orthologs or paralogs.

Substantially homologous parent proteins and polypeptides arecharacterized as having one or more amino acid substitutions, deletionsor additions. These changes are preferably of a minor nature, that isconservative amino acid substitutions (see Table 1) and othersubstitutions that do not significantly affect the folding or activityof the protein or polypeptide; small deletions, typically of one toabout 30 amino acids; and small amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or a small extension that facilitatespurification (an affinity tag), such as a poly-histidine tract, proteinA (Nilsson et al., EMBO J. 4:1075, 1985; Nilsson et al., MethodsEnzymol. 198:3, 1991. See, in general Ford et al., Protein Expressionand Purification 2: 95-107, 1991, which is incorporated herein byreference. DNAs encoding affinity tags are available from commercialsuppliers (e.g., Pharmacia Biotech, Piscataway, N.J.; New EnglandBiolabs, Beverly, Mass.).

However, even though the changes described above preferably are of aminor nature, such changes may also be of a larger nature such as fusionof larger polypeptides of up to 300 amino acids or more both as amino-or carboxyl-terminal extensions. TABLE 1 Conservative amino acidsubstitutions Basic: arginine lysine Acidic: glutamic acid aspartic acidPolar: glutamine asparagines serine threonine cysteine Hydrophobic:leucine isoleucine valine proline methionine Aromatic/ phenylalanineHeteroaromatic: tryptophan tyrosine histidine Small: glycine alanine

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline and a-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, or preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.Essential amino acids in the pectate lyase polypeptides of the presentinvention can be identified according to procedures known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244: 1081-1085, 1989). In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (i.e pectate lyase activity) to identify amino acidresidues that are critical to the activity of the molecule. See also,Hilton et al., J. Biol. Chem. 271:4699-4708, 1996. The active site ofthe enzyme or other biological interaction can also be determined byphysical analysis of structure, as determined by such techniques asnuclear magnetic resonance, crystallography, electron diffraction orphotoaffinity labeling, in conjunction with mutation of putative contactsite amino acids. See, for example, de Vos et al., Science 255:306-312,1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al.,FEBS Lett. 309:59-64, 1992. The identities of essential amino acids canalso be inferred from analysis of homologies with polypeptides which arerelated to a polypeptide according to the invention.

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis, recombination and/or shuffling followed by arelevant screening procedure, such as those disclosed by Reidhaar-Olsonand Sauer (Science 241:53-57, 1988), Bowie and Sauer (Proc. Natl. Acad.Sci. USA 86:2152-2156, 1989), WO95/17413, or WO 95/22625. Briefly, theseauthors disclose methods for simultaneously randomizing two or morepositions in a polypeptide, or recombination/shuffling of differentmutations (WO95/17413, WO95/22625), followed by selecting for functionala polypeptide, and then sequencing the mutagenized polypeptides todetermine the spectrum of allowable substitutions at each position.Other methods that can be used include phage display (e.g., Lowman etal., Biochem. 30:10832-10837, 1991; Ladner et al., U.S. Pat. No.5,223,409; Huse, WIPO Publication WO 92/06204) and region-directedmutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA7:127, 1988).

Mutagenesis/shuffling methods as disclosed above can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode active polypeptides can be recovered from the hostcells and rapidly sequenced using modern equipment. These methods allowthe rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure. Using the methods discussed above,one of ordinary skill in the art can identify and/or prepare a varietyof polypeptides that are substantially homologous to residues 1 to 399of SEQ ID NO: 2 and retain the pectate lyase activity of the parentenzyme.

However, the very same methods are also useful for providing the pectatelyase variants of the invention having more advantageous properties thanthe wild-type protein. Using these methods, the present inventors haveidentified a number of positions in which the wild-type pectate lyase ofSEQ ID NO:2 may advantageously by substituted in order to preparevariants with improved properties.

Preferred pectate lyase variants of the inventions are substituted inone or more of the following positions (numbering relative to SEQ IDNO:2): 5, 9, 11, 26, 28, 30, 31, 37, 40, 45, 46, 47, 48, 49, 50, 51, 52,54, 61, 64, 68, 69, 70, 71, 74, 75, 76, 79, 86, 87, 91, 99, 105, 106,107, 111, 115, 116, 118, 122, 123, 134, 136, 139, 140, 141, 146, 148,156, 158, 170, 182, 185, 186, 189, 193, 194, 196, 199, 201, 202, 204,213, 215, 218, 224, 228, 229, 234, 235, 237, 251, 256, 257, 258, 272,277, 286, 295, 298, 301, 302, 303, 305, 307, 308, 314, 316, 323, 324,326, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 349, 356,357, 363, 366, 378, 381, 384, 386, 387, 389, 390, 391, 393 and 397.

Preferred variants of the present invention further comprise variants inwhich the overall charge of the enzyme has been made more negative. Insuch variants positively charged amino acids may have been replacedand/or amino acids, which are negatively charged under the applicationconditions, have been introduced.

Thus, in accordance herewith, preferred variants may have had replacedan amino acid residue being partly or fully positively charged underapplication conditions, i.e. a His, Lys or an Arg. Further, preferredvariants may have had replaced any residue by an amino acid residue witha negative charge under application conditions, i.e. Asp, Glu, and Tyr.

Especially preferred variants are those in which a lysine residue in oneor more of the following positions (numbering relative to SEQ ID NO:2):26, 47, 54, 59, 71, 79, 87, 90, 99, 100, 115, 118, 139, 148, 213, 218,247, 257, 263, 265, 274, 314, 317, 334, 386 and 397, has been replaced.

Likewise, especially preferred variants are those in which an arginineresidue in one or more of the following positions (numbering relative toSEQ ID NO:2): 38, 110, 112, 120, 155, 206, 217, 272, 279, 282 and 284,has been replaced.

Further, preferred variants are also those in which a histidine residuein one or more of the following positions (numbering relative to SEQ IDNO:2): 5, 31, 193, 198, 221, 222, 243, 245, 269, 270, 289, 376 and 384,has been replaced.

Preferred variant pectate lyase enzymes have been modified in order tochange the binding constant for Ca2+, and thereby improving thecalcium-depletion stability. In a pectate lyase of the presentinvention, the three amino acid residues D184, D223 and D227 arecoordinating the binding of Ca2+ at the primary calcium binding site. Byrecruiting a fourth amino acid residue in this coordination it ispossible to change the binding constant for Ca2+. The presence of Ca2+at the primary calcium binding site may influence either the catalyticevent (by reducing the pKa of the catalytic residue), the alignment ofthe substrate or determine whether the enzyme functions as a hydrolaseor a lyase (the presence of Ca2+ being a prerequisite for lyaseactivity). Such variants with improved calcium-depletion stability maycomprise one of the substitutions Q182D and Q182E.

Further examples of preferred variants are those with improved oxidationstability in which an oxidation labile amino acid residue has beenreplaced. By “oxidation labile” are meant amino acids holding a sulphur-or a hydroxyl-group, e.g. methionine, cysteine, threonine, serine andtyrosine. Preferred variants are those in which an oxidation labileamino acid residue in one or more of the following positions (numberingrelative to SEQ ID NO:2): 64, 122, 199 and 237 have been replaced.

Further examples of preferred variants are those holding an amino acidsubstitution in a flexible residue, wherein a less flexible amino acidresidue has been introduced. In the present context the term “flexible”refers to the number of possible phi and psi angles of the C-alpha atomin the amino acid. The flexibility of the peptide backbone is limited bysteric hindrance of the atoms bound to the C-alpha atom. In general onlythe amino acid side chain differs from one residue to the other, thusthe size of the side chain (the Van der Waals radius) determines theflexibility. A glycine has the smallest side chain, a hydrogen atom;therefore a glycine residue introduces more flexibility. The oppositesituation applies for proline, where the possible conformations arelimited not only by a large side chain but also due to the ringstructure. Thus a proline residue is less flexible than average, andgives stiffness and stability to the peptide.

Such less flexible variants include in particular, but are not limitedto, variants in which a glycine residue have been substituted with anyof the other 19 naturally occurring amino acids or variants in which anyamino acid have been substituted with a proline residue. In especiallypreferred variants a less flexible amino acid residue has beenintroduced in one or more of the following regions (numbering ofpositions relative to SEQ ID NO:2): 26-31, 45-50, 66-72, 81-89, 90-106,134-137, 169-178, 210-217, 253-262, 275-286, 297-308, 328-343, 354-356,361-365, 368-372 and 376-379.

In a preferred embodiment of the present invention, the Bacillussubtilis pectate lyase variant comprises at least one substituted aminoacid residue selected from the group consisting of: H5R, E9G, N11Y,K26Q, S28T, S30F, S30P, S30T, H31N, N37D, Q40E, L45V, G46D. K47N, K47R,D48E, D48P, T49P, N50D, N50L, N50Y, N51Y, T52M, K54V, T61A, M64F, D68*,N69*, L70*, K71*, K71E, G74D, L75A, L75P, N76D, K79A, D86N, K87A, K87E,A91E, K99I, K99N, K99R, T105A, T105P, L106Q, E107K, A111E, K115A, K115I,K115N, K115Q, N116D, K118A, K118E, M122E, M122K, M122N, M122Q, V123l,S134L, T136S, K139E, K139F, K139I, K139M, K139N, K139S, I140V, V141G,V141E, V141L, V141N, Q146F, Q146H, Q146I, Q146V, K148E, K148Q, N156S,E158N, D170N, Q182D, Q182E, N185H, N186H, N189D, H193Y, I194V, I196V,C199N, C199S, F201L, N202K, G204R, K213E, K213N, K213T, F215Y, K218E,K218L, K218P, G224S, A228I, S229T, Y234H, I235V, M237I, S256C, K257E,K257N, T258I, L286Y, R272C, R272H, R272Y, V277D, G286A, Y295H, S298N,S301Y, S302A, D303S, A305P, S307R, Y308S, K314N, S316F, N323M, V324A,D326N, S331P, S331T, A332P, A333E, K334E, T335S, T335R, I336S, S337C,S337K, S337L, S337R, V338E, V338Y, F339I, S340A, S340K, S340N, S340P,S340Q, G341S, G349R, Q356H, I357V, N363S, S366N, T378G, T378S, A381D,H384N, K386P, K386R, S387A, V389I, I390N, I390T, S391N, A393V and K397D.

It is at present contemplated that one or more of these substitutionseither alone or in combination increase the detergent stability of thepectate lyase variant when compared to the parent enzyme.

Preferred multiple substitutions which increase the detergent stabilityinclude:

-   A228I+F251I,-   S134L+K257E,-   K115I+K213E,-   K139I+K213N,-   H5R+K257N+S302A,-   K99I+I196V,-   K115A+K118A,-   K115A+K118A+M122N,-   V141E+C199S+K213E,-   K115I+Q146H,-   K71E+K118E,-   T49P+N156S,-   K314N+S340P,-   V141E+I235V,-   G46D+K257N,-   S28T+S30F+K334E+N363S,-   D48E+L106Q+I140V+F215Y+K218E,-   H193Y+S256C+V389I+A393V,-   E9G+H31N+N50D+L106Q+A111E+T136S+V141L+F201L+N202K+F215Y+G286A+A381D+H384N,-   K213N+T258I,-   E9G+H31N+L106Q+D303S+A305P+T335S+H384N+S391N,-   E9G+H31N+D48E+L106Q+A111E+S301Y+D303S+A305P+T378S+H384N+S391N,-   L45V+N50Y+N185H,-   N11Y+K87E+K99N,-   E9G+D48E+L106Q+S316F+A381D,-   S30P+K115I+K139I+Q146H+S337C,-   E9G+H31N+D48E+L106Q+I140V+F215Y+D303S+A305P+T378S+H384N+S391N,-   H31N+T105A+L106Q+A111E+V141L+K218E+D303S+A305P+D326N+T335S+H384N+S391N,-   K26Q+K47N+L106Q+I140V+F215Y+D303S+A305P+T378S+H384N+S391N,-   D48E+L106Q+I140V+F215Y+D303S+A305P+T378S+H384N+S391N,-   K213T+K218L+A305P,-   M64F+K213T+K218L+A305P,-   M64F+M122K+K118E+K213T+K218L+A305P,-   K139I+Q146H+K257N+S337C,-   M64F+K139I+Q146H+S337C,-   K139I+Q146H+S337C and-   D48P+M64F+T105P+K139I+Q146H+K213T+K218P+T258I+A305P+S331P+S337K.    Protein Production

The polypeptides of the present invention, including full-lengthproteins, fragments thereof and fusion proteins, can be produced ingenetically engineered host cells according to conventional techniques.Suitable host cells are those cell types that can be transformed ortransfected with exogenous DNA and grown in culture, and includebacteria, fungal cells, and cultured higher eukaryotic cells. Bacterialcells, particularly cultured cells of gram-positive organisms, arepreferred. Gram-positive cells from the genus of Bacillus are especiallypreferred, such as B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans,B. circulans, B. lautus, B. thuringiensis, B. agaradherens, or inparticular B. subtilis.

Techniques for manipulating cloned DNA molecules and introducingexogenous DNA into a variety of host cells are disclosed by Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989; Ausubel et al. (eds.),Current Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y.,1987; and (Bacillus subtilis and Other Gram-Positive Bacteria,Sonensheim et al., 1993, American Society for Microbiology, WashingtonD.C.), which are incorporated herein by reference.

In general, a DNA sequence encoding a pectate lyase of the presentinvention is operably linked to other genetic elements required for itsexpression, generally including a transcription promoter and terminatorwithin an expression vector. The vector will also commonly contain oneor more selectable markers and one or more origins of replication,although those skilled in the art will recognize that within certainsystems selectable markers may be provided on separate vectors, andreplication of the exogenous DNA may be provided by integration into thehost cell genome. Selection of promoters, terminators, selectablemarkers, vectors and other elements is a matter of routine design withinthe level of ordinary skill in the art. Many such elements are describedin the literature and are available through commercial suppliers.

To direct a polypeptide into the secretory pathway of a host cell, asecretory signal sequence (also known as a leader sequence, preprosequence or pre sequence) is provided in the expression vector. Thesecretory signal sequence may be that of the polypeptide, or may bederived from another secreted protein or synthesized de novo. Numeroussuitable secretory signal sequences are known in the art and referenceis made to Bacillus subtilis and Other Gram-Positive Bacteria,Sonenshein et al., 1993, (American Society for Microbiology, WashingtonD.C.); and Cutting, S. M.(eds.) “Molecular Biological Methods forBacillus”, (John Wiley and Sons, 1990) for further description ofsuitable secretory signal sequences especially for secretion in aBacillus host cell. The secretory signal sequence is joined to the DNAsequence in the correct reading frame. Secretory signal sequences arecommonly positioned 5′ to the DNA sequence encoding the polypeptide ofinterest, although certain signal sequences may be positioned elsewherein the DNA sequence of interest (see, e.g., Welch et al., U.S. Pat. No.5,037,743; Holland et al., U.S. Pat. No. 5,143,830). Transformed ortransfected host cells are cultured according to conventional proceduresin a culture medium containing nutrients and other components requiredfor the growth of the chosen host cells. A variety of suitable media,including defined media and complex media, are known in the art andgenerally include a carbon source, a nitrogen source, essential aminoacids, vitamins and minerals. Media may also contain such components asgrowth factors or serum, as required. The growth medium will generallyselect for cells containing the exogenously added DNA by, for example,drug selection or deficiency in an essential nutrient which iscomplemented by the selectable marker carried on the expression vectoror co-transfected into the host cell.

The fermentation may be carried out by cultivation of the host cellunder aerobic conditions in a nutrient medium containing carbon andnitrogen sources together with other essential nutrients, the mediumbeing composed in accordance with the principles of the known art. Themedium may be a complex rich medium or a minimal medium. The nitrogensource may be of inorganic and/or organic nature. Suitable inorganicnitrogen sources are nitrates and ammonium salts. Among the organicnitrogen sources quite a number are used regularly in fermentations.Examples are soybean meal, casein, corn, corn steep liquor, yeastextract, urea and albumin. Suitable carbon sources are carbohydrates orcarbohydrate containing materials. Preferably the nutrient mediumcontains pectate, polygalacturonic acid and/or pectin esterified to ahigher or lower degree as carbon source and/or inducer of pectinaseproduction. Alternatively, the medium contains a pectin rich materialsuch as soybean meal, apple pulp or citrus peel.

The cultivation may preferably be conducted at alkaline pH values suchas at least pH 8 or at least pH 9, which can be obtained by addition ofsuitable buffers such as sodium carbonate or mixtures of sodiumcarbonate and sodium bicarbonate after sterilisation of the growthmedium.

Protein Isolation

When the expressed recombinant polypeptide is secreted the polypeptidemay be purified from the growth media. Preferably the expression hostcells are removed from the media before purification of the polypeptide(e.g. by centrifugation).

When the expressed recombinant polypeptide is not secreted from the hostcell, the host cell are preferably disrupted and the polypeptidereleased into an aqueous “extract” which is the first stage of suchpurification techniques. Preferably the expression host cells areremoved from the media before the cell disruption (e.g. bycentrifugation).

The cell disruption may be performed by conventional techniques such asby lysozyme digestion or by forcing the cells through high pressure. See(Robert K. Scobes, Protein Purification, Second edition,Springer-Verlag) for further description of such cell disruptiontechniques.

Whether or not the expressed recombinant polypeptides (or chimericpolypeptides) is secreted or not it can be purified using fractionationand/or conventional purification methods and media.

Ammonium sulfate precipitation and acid or chaotrope extraction may beused for fractionation of samples. Exemplary purification steps mayinclude hydroxyapatite, size exclusion, FPLC and reverse-phase highperformance liquid chromatography. Suitable anion exchange media includederivatized dextrans, agarose, cellulose, polyacrylamide, specialtysilicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred,with Fast-Flow Sepharose (Pharmacia Biotech, Piscataway, N.J.) beingparticularly preferred. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties. Examples of coupling chemistriesinclude cyanogen bromide activation, N-hydroxysuccinimide activation,epoxide activation, sulfhydryl activation, hydrazide activation, andcarboxyl and amino derivatives for carbodiimide coupling chemistries.These and other solid media are well known and widely used in the art,and are available from commercial suppliers.

Selection of a particular method is a matter of routine design and isdetermined in part by the properties of the chosen support. See, forexample, Affinity Chromatography: Principles & Methods, Pharmacia LKBBiotechnology, Uppsala, Sweden, 1988.

Polypeptides of the invention or fragments thereof may also be preparedthrough chemical synthesis. Polypeptides of the invention may bemonomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; and may or may not include an initial methionine aminoacid residue.

Accordingly, in a further aspect, the present invention also relates toa method of producing the enzyme preparation of the invention, themethod comprising culturing a microorganism capable of producing thepectate lyase variant under conditions permitting the production of theenzyme, and recovering the enzyme from the culture. Culturing may becarried out using conventional fermentation techniques, e.g. culturingin shake flasks or fermentors with agitation to ensure sufficientaeration on a growth medium inducing production of the pectate lyasevariant. The growth medium may contain a conventional N-source such aspeptone, yeast extract or casamino acids, a reduced amount of aconventional C-source such as dextrose or sucrose, and an inducer suchas pectate or pectin or composit plant substrates such as cereal brans(e.g. wheat bran or rice husk). The recovery may be carried out usingconventional techniques, e.g. separation of bio-mass and supernatant bycentrifugation or filtration, recovery of the supernatant or disruptionof cells if the enzyme of interest is intracellular, perhaps followed byfurther purification as described in EP 0 406 314 or by crystallizationas described in WO 97/15660.

In yet another aspect, the present invention relates to an isolatedpectate lyase variant having the properties described above and which isfree from homologous impurities, and is produced using conventionalrecombinant techniques.

Methods and Uses

Microtiter assay for Quantification of Pectate Lyase Activity

Pectate lyase cleaves polygalacturonic acid through a trans eliminationmechanism. This means that it leaves a double C—C bond for eachsubstrate split. This bond absorbs at 235 nm allowing direct detectionof pectate lyase action on soluble polygalacturonic acid by measuringabsorbance at that wavelength.

An enzyme sample is diluted in assay buffer (100 mM Tris-HCl, 0.68 mMCaCl₂, pH 8.0) to a concentration between 5 and 100 ng/ml. If the enzymesample contains detergent it should be diluted at least a 1000-fold withrespect to detergent.

100 μl of the enzyme buffer dilution is mixed with 100 μl substrate (1%(w/v) polygalacturonic acid from Sigma, P-3850, stirred in assay bufferfor at least 15 min and centrifuged for 5 min at 2300 g, supernatant isused.) in a heating plate and heated to 40° C. for 10 min. in a heatingblock, preferably a PCR machine or equipment of equivalent accuracy andheating speeds.

100 μl enzyme/substrate solution is mixed with 100 μl stop reagent (50mM H₃PO₄) in a UV-transparent microtiter plate. The UV plate is shakenbriefly and gently, and the absorbance at 235 nm is measured in amicrotiter spectrometer (Molecular Devices, SpectraMAX 190). Theabsorbance readings are corrected for background absorbance bysubtracting the absorbance of a control sample, run without enzymeadded, to all measured values.

A standard curve based on the activity of the pectate lyase of SEQ IDNO:2 (from Bacillus subtilis deposited as IFO 3134) was linear between2.5 and 100 ng/ml enzyme in the reaction mixture: Absorbance at 235 nm(AU), Enzyme dose (ng/ml) background subtracted 0 0.00 2.5 0.03 5 0.0710 0.16 15 0.26 25 0.42 50 0.85 100 1.83

Alternatively, catalytic activity of pectate lyase can be determined bythe viscosity assay, APSU.

Viscosity Assay, APSU

APSU units: The APSU assay measures the change in viscosity of asolution of polygalacturonic acid in the absence of added calcium ions.

A 5% w/v solution of sodium polygalacturonate (Sigma P-1879) issolubilised in 0.1 M glycine buffer, pH 10.4 ml of this solution arepreincubated for 5 min at 40 degrees Celsius. Then, 250 microlitre ofthe enzyme (or enzyme dilution) are added, after which the reaction ismixed for 10 sec on a mixer at the highest speed and incubated for 20min at 40 degrees Celsius or at another temperature.

Viscosity is measured using a MIVI 600 viscometer (Sofraser, 45700Villemandeur, France). Viscosity is measured as mV after 10 sec. Forcalculation of APSU units the following standard curve is used: APSU/ml0.00 4.00 9.00 14.00 19.00 24.00 34.00 49.00 99.00 mV 300 276 249 227206 188 177 163 168Use in the Detergent Industry

In further aspects, the present invention relates to a detergentcomposition comprising the pectate lyase variant or pectate lyasevariant preparation of the invention, and to a process for machinetreatment of fabrics comprising treating fabric during a washing cycleof a machine washing process with a washing solution containing thepectate lyase variant or pectate lyase variant preparation of theinvention.

Typically, the detergent composition of the invention comprisesconventional ingredients such as surfactants (anionic, nonionic,zwitterionic, amphoteric), builders, and other ingredients, e.g. asdescribed in WO 97/01629 which is hereby incorporated by reference inits entirety.

Use in the Textile and Cellulosic Fiber Processing Industries

The pectate lyase variant of the present invention can be used incombination with other carbohydrate-degrading enzymes (for instancehemicellulases, such as arabinanase, xyloglucanase, mannanase andpectinase) for biopreparation of fibers or for cleaning of fibers incombination with detergents. Cotton fibers consist of a primary cellwall layer containing pectin and a secondary layer containing mainlycellulose. Under cotton preparation or cotton refining part of theprimary cell wall will be removed. The present invention relates toeither help during cotton refining by removal of the primary cell wall,or during cleaning of the cotton to remove residual pectic substancesand prevent graying of the textile.

In the present context, the term “cellulosic material” is intended tomean fibers, sewn and unsewn fabrics, including knits, wovens, denims,yarns, and toweling, made from cotton, cotton blends or natural ormanmade cellulosics (e.g. originating from xylan-containing cellulosefibers such as from wood pulp) or blends thereof. Examples of blends areblends of cotton or rayon/viscose with one or more companion materialsuch as wool, synthetic fibers (e.g. polyamide fibers, acrylic fibers,polyester fibers, polyvinyl alcohol fibers, polyvinyl chloride fibers,polyvinylidene chloride fibers, polyurethane fibers, polyurea fibers,aramid fibers), and cellulose-containing fibers (e.g. rayon/viscose,ramie, hemp, flax/linen, jute, cellulose acetate fibers, lyocell).

The preparation of the present invention is useful in the cellulosicfiber processing industry for the pre-treatment or retting of fibersfrom hemp, flax or linen.

The processing of cellulosic material for the textile industry, as forexample cotton fiber, into a material ready for garment manufactureinvolves several steps: spinning of the fiber into a yarn; constructionof woven or knit fabric from the yarn and subsequent preparation, dyeingand finishing operations. Woven goods are constructed by weaving afilling yarn between a series of warp yarns; the yarns could be twodifferent types. Knitted goods are constructed by forming a network ofinterlocking loops from one continuous length of yarn. The cellulosicfibers can also be used for non-woven fabric.

The preparation process prepares the textile for the proper response indyeing operations. The sub-steps involved in preparation are

a. Desizing (for woven goods) using polymeric size like e.g. starch, CMCor PVA is added before weaving in order to increase the warp speed; Thismaterial must be removed before further processing.

b. Scouring, the aim of which is to remove non-cellulosic material fromthe cotton fiber, especially the cuticle (mainly consisting of waxes)and primary cell wall (mainly consisting of pectin, protein andxyloglucan). A proper wax removal is necessary for obtaining a highwettability, being a measure for obtaining a good dyeing. Removal of theprimary cell wall—especially the pectins—improves wax removal andensures a more even dyeing. Further this improves the whiteness in thebleaching process. The main chemical used in scouring is sodiumhydroxide in high concentrations, up to 70 g/kg cotton and at hightemperatures, 80-95 degrees Celsius; and

c. Bleaching; normally the scouring is followed by a bleach usinghydrogen peroxide as the oxidizing agent in order to obtain either afully bleached (white) fabric or to ensure a clean shade of the dye.

A one step combined scour/bleach process is also used by the industry.Although preparation processes are most commonly employed in the fabricstate; scouring, bleaching and dyeing operations can also be done at thefiber or yarn stage.

The processing regime can be either batch or continuous with the fabricbeing contacted by the liquid processing stream in open width or ropeform. Continuous operations generally use a saturator whereby anapproximate equal weight of chemical bath per weight of fabric isapplied to the fabric, followed by a heated dwell chamber where thechemical reaction takes place. A washing section then prepares thefabric for the next processing step. Batch processing generally takesplace in one processing bath whereby the fabric is contacted withapproximately 8-15 times its weight in chemical bath. After a reactionperiod, the chemicals are drained, fabric rinsed and the next chemicalis applied. Discontinuous pad-batch processing involves a saturatorwhereby an approximate equal weight of chemical bath per weight offabric is applied to the fabric, followed by a dwell period, which, inthe case of cold pad-batch, might be one or more days.

Woven goods are the prevalent form of textile fabric construction. Theweaving process demands a “sizing” of the warp yarn to protect it fromabrasion. Starch, polyvinyl alcohol (PVA), carboxymethyl cellulose,waxes and acrylic binders are examples of typical sizing chemicals usedbecause of availability and cost. The size must be removed after theweaving process as the first step in preparing the woven goods. Thesized fabric in either rope or open width form is brought in contactwith the processing liquid containing the desizing agents. The desizingagent employed depends upon the type of size to be removed. For PVAsizes, hot water or oxidative processes are often used. The most commonsizing agent for cotton fabric is based upon starch. Therefore mostoften, woven cotton fabrics are desized by a combination of hot water,the enzyme alfa-amylase to hydrolyze the starch and a wetting agent orsurfactant. The cellulosic material is allowed to stand with thedesizing chemicals for a “holding period” sufficiently long toaccomplish the desizing. The holding period is dependent upon the typeof processing regime and the temperature and can vary from 15 minutes to2 hours, or in some cases, several days. Typically, the desizingchemicals are applied in a saturator bath which generally ranges fromabout 15 degrees Celsius to about 55 degrees Celsius. The fabric is thenheld in equipment such as a “J-box” which provides sufficient heat,usually between about 55 degrees Celsius and about 100 degrees Celsius,to enhance the activity of the desizing agents. The chemicals, includingthe removed sizing agents, are washed away from the fabric after thetermination of the holding period. In order to ensure a high whitenessor a good wettability and resulting dyeability, the size chemicals andother applied chemicals must be thoroughly removed. It is generallybelieved that an efficient desizing is of crucial importance to thefollowing preparation processes: scouring and bleaching.

The scouring process removes much of the non-cellulosic compoundsnaturally found in cotton. In addition to the natural non-cellulosicimpurities, scouring can remove dirt, soils and residual manufacturingintroduced materials such as spinning, coning or slashing lubricants.The scouring process employs sodium hydroxide or related causticizingagents such as sodium carbonate, potassium hydroxide or mixturesthereof. Generally an alkali stable surfactant is added to the processto enhance solubilization of hydrophobic compounds and/or prevent theirredeposition back on the fabric. The treatment is generally at a hightemperature, 80-100 degrees Celsius, employing strongly alkalinesolutions, pH 13-14, of the scouring agent. Due to the non-specificnature of chemical processes not only are the impurities but thecellulose itself is attacked, leading to damages in strength or otherdesirable fabric properties. The softness of the cellulosic fabric is afunction of residual natural cotton waxes. The non-specific nature ofthe high temperature strongly alkaline scouring process cannotdiscriminate between the desirable natural cotton lubricants and themanufacturing introduced lubricants. Furthermore, the conventionalscouring process can cause environmental problems due to the highlyalkaline effluent from these processes. The scouring stage prepares thefabric for the optimal response in bleaching. An inadequately scouredfabric will need a higher level of bleach chemical in the subsequentbleaching stages. The bleaching step decolorizes the natural cottonpigments and removes any residual natural woody cotton trash componentsnot completely removed during ginning, carding or scouring. The mainprocess in use today is an alkaline hydrogen peroxide bleach. In manycases, especially when a very high whiteness is not needed, bleachingcan be combined with scouring.

In the examples below it is shown that the scouring step can be carriedout using the pectate lyase or pectate lyase preparation of the presentinvention a temperature of about 50-80 degrees Celsius and a pH of about7-11, thus substituting or supplementing the highly causticizing agents.An optimized enzymatic process ensures a high pectin removal and fullwettability.

Degradation or Modification of Plant Material

The enzyme or enzyme preparation according to the invention ispreferably used as an agent for degradation or modification of plantcell walls or any pectin-containing material originating from plantcells walls due to the high plant cell wall degrading activity of thepectate lyase variant of the invention.

The pectate lyase variant of the present invention may be used alone ortogether with other enzymes like glucanases, pecfinases and/orhemicellulases to improve the extraction of oil from oil-rich plantmaterial, like soy-bean oil from soy-beans, olive-oil from olives orrapeseed-oil from rape-seed or sunflower oil from sunflower.

The pectate lyase variant of the present invention may be used forseparation of components of plant cell materials. Of particular interestis the separation of sugar or starch rich plant material into componentsof considerable commercial interest (like sucrose from sugar beet orstarch from potato) and components of low interest (like pulp or hullfractions). Also, of particular interest is the separation ofprotein-rich or oil-rich crops into valuable protein and oil andinvaluable hull fractions. The separation process may be performed byuse of methods known in the art.

The pectate lyase variant of the invention may also be used in thepreparation of fruit or vegetable juice in order to increase yield, andin the enzymatic hydrolysis of various plant cell wall-derived materialsor waste materials, e.g. from wine or juice production, or agriculturalresidues such as vegetable hulls, bean hulls, sugar beet pulp, olivepulp, potato pulp, and the like.

The plant material may be degraded in order to improve different kindsof processing, facilitate purification or extraction of other componentthan the galactans like purification of pectins from citrus, improve thefeed value, decrease the water binding capacity, improve thedegradability in waste water plants, improve the conversion of plantmaterial to ensilage, etc.

By means of an enzyme preparation of the invention it is possible toregulate the consistency and appearance of processed fruit orvegetables. The consistency and appearance has been shown to be aproduct of the actual combination of enzymes used for processing, i.e.the specificity of the enzymes with which the pectate lyase variant ofthe invention is combined. Examples include the production of clearjuice e.g. from apples, pears or berries; cloud stable juice e.g. fromapples, pears, berries, citrus or tomatoes; and purees e.g. from carrotsand tomatoes.

The pectate lyase variant of the invention may be used in modifying theviscosity of plant cell wall derived material. For instance, the pectatelyase variant may be used to reduce the viscosity of feed containinggalactan and to promote processing of viscous galactan containingmaterial. The viscosity reduction may be obtained by treating thegalactan containing plant material with an enzyme preparation of theinvention under suitable conditions for full or partial degradation ofthe galactan containing material The pectate lyase variant can be usede.g. in combination with other enzymes for the removal of pecticsubstances from plant fibres. This removal is essential e.g. in theproduction of textile fibres or other cellulosic materials. For thispurpose plant fibre material is treated with a suitable amount of thepectate lyase of the invention under suitable conditions for obtainingfull or partial degradation of pectic substances associated with theplant fibre material.

Animal Feed Additive

Pectate lyase variants of the present invention may be used formodification of animal feed and may exert their effect either in vitro(by modifying components of the feed) or in vivo. The pectate lyasevariant is particularly suited for addition to animal feed compositionscontaining high amounts of arabinogalactans or galactans, e.g. feedcontaining plant material from soy bean, rape seed, lupin etc. Whenadded to the feed the pectate lyase variant significantly improves thein vivo break-down of plant cell wall material, whereby a betterutilization of the plant nutrients by the animal is achieved. Thereby,the growth rate and/or feed conversion ratio (i.e. the weight ofingested feed relative to weight gain) of the animal is improved. Forexample the indigestible galactan is degraded by pectate lyase, e.g. incombination with beta-galactosidase, to galactose or galactooligomerswhich are digestible by the animal and thus contribute to the availableenergy of the feed. Also, by the degradation of galactan the pectatelyase may improve the digestibility and uptake of non-carbohydrate feedconstituents such as protein, fat and minerals.

For further description reference is made to PCT/DK 96/00443 and aworking example herein.

Wine and Juice Processing

The enzyme or enzyme preparation of the invention may be used forde-pectinization and viscosity reduction in vegetable or fruit juice,especially in apple or pear juice. This may be accomplished by treatingthe fruit or vegetable juice with an enzyme preparation of the inventionin an amount effective for degrading pectin-containing materialcontained in the fruit or vegetable juice.

The enzyme or enzyme preparation may be used in the treatment of mashfrom fruits and vegetables in order to improve the extractability ordegradability of the mash. For instance, the enzyme preparation may beused in the treatment of mash from apples and pears for juiceproduction, and in the mash treatment of grapes for wine production.

The applicability of pectate lyase variants with improved detergentstability is appreciated whenever the variants are used in anenvironment comprising surfactants.

DETERGENT DISCLOSURE AND EXAMPLES

Surfactant System

The detergent compositions according to the present invention comprise asurfactant system, wherein the surfactant can be selected from nonionicand/or anionic and/or cationic and/or ampholytic and/or zwitterionicand/or semi-polar surfactants.

The surfactant is typically present at a level from 0.1% to 60% byweight.

The surfactant is preferably formulated to be compatible with enzymecomponents present in the composition. In liquid or gel compositions thesurfactant is most preferably formulated in such a way that it promotes,or at least does not degrade, the stability of any enzyme in thesecompositions.

Preferred systems to be used according to the present invention compriseas a surfactant one or more of the nonionic and/or anionic surfactantsdescribed herein.

Polyethylene, polypropylene, and polybutylene oxide conden-sates ofalkyl phenols are suitable for use as the nonionic surfactant of thesurfactant systems of the present invention, with the polyethylene oxidecondensates being preferred. These compounds include the condensationproducts of alkyl phenols having an alkyl group containing from about 6to about 14 carbon atoms, preferably from about 8 to about 14 carbonatoms, in either a straight chain or branched-chain configuration withthe alkylene oxide. In a preferred embodiment, the ethylene oxide ispresent in an amount equal to from about 2 to about 25 moles, morepreferably from about 3 to about 15 moles, of ethylene oxide per mole ofalkyl phenol. Commercially available nonionic surfactants of this typeinclude Igepal™ CO-630, marketed by the GAF Corporation; and Triton™X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas Company.These surfactants are commonly referred to as alkylphenol alkoxylates(e.g., alkyl phenol ethoxylates).

The condensation products of primary and secondary aliphatic alcoholswith about 1 to about 25 moles of ethylene oxide are suitable for use asthe nonionic surfactant of the nonionic surfactant systems of thepresent invention. The alkyl chain of the aliphatic alcohol can eitherbe straight or branched, primary or secondary, and generally containsfrom about 8 to about 22 carbon atoms. Preferred are the condensationproducts of alcohols having an alkyl group containing from about 8 toabout 20 carbon atoms, more preferably from about 10 to about 18 carbonatoms, with from about 2 to about 10 moles of ethylene oxide per mole ofalcohol. About 2 to about 7 moles of ethylene oxide and most preferablyfrom 2 to 5 moles of ethylene oxide per mole of alcohol are present insaid condensation products. Examples of commercially available nonionicsurfactants of this type include Tergitol™ 15-S-9 (The condensationproduct of C₁₁-C₁₅ linear alcohol with 9 moles ethylene oxide),Tergitol™ 24-L-6 NMW (the condensation product of C₁₂-C₁₄ primaryalcohol with 6 moles ethylene oxide with a narrow molecular weightdistribution), both marketed by Union Carbide Corporation; Neodol™ 45-9(the condensation product of C₁₄-C₁₅ linear alcohol with 9 moles ofethylene oxide), Neodol™ 23-3 (the condensation product of C₁₂-C₁₃linear alcohol with 3.0 moles of ethylene oxide), Neodol™ 45-7 (thecondensation product of C₁₄-C₁₅ linear alcohol with 7 moles of ethyleneoxide), Neodol™ 45-5 (the condensation product of C₁₄-C₁₅ linear alcoholwith 5 moles of ethylene oxide) marketed by Shell Chemical Company,Kyro™ EOB (the condensation product of C₁₃-C₁₅ alcohol with 9 molesethylene oxide), marketed by The Procter & Gamble Company, and GenapolLA 050 (the condensation product of C₁₂-C₁₄ alcohol with 5 moles ofethylene oxide) marketed by Hoechst. Preferred range of HLB in theseproducts is from 8-11 and most preferred from 8-10.

Also useful as the nonionic surfactant of the surfactant systems of thepresent invention are alkylpolysaccharides disclosed in U.S. Pat. No.4,565,647, having a hydrophobic group containing from about 6 to about30 carbon atoms, preferably from about 10 to about 16 carbon atoms and apolysaccharide, e.g. a polyglycoside, hydrophilic group containing fromabout 1.3 to about 10, preferably from about 1.3 to about 3, mostpreferably from about 1.3 to about 2.7 saccharide units. Any reducingsaccharide containing 5 or 6 carbon atoms can be used, e.g., glucose,galactose and galactosyl moieties can be substituted for the glucosylmoieties (optionally the hydrophobic group is attached at the 2-, 3-,4-, etc. positions thus giving a glucose or galactose as opposed to aglucoside or galactoside). The intersaccharide bonds can be, e.g.,between the one position of the additional saccharide units and the 2-,3-, 4-, and/or 6-positions on the preceding saccharide units.

The preferred alkylpolyglycosides have the formulaR²O(C_(n)H_(2n)O)_(t)(glycosyl)_(x)wherein R² is selected from the group consisting of alkyl, alkylphenyl,hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof in which thealkyl groups contain from about 10 to about 18, preferably from about 12to about 14, carbon atoms; n is 2 or 3, preferably 2; t is from 0 toabout 10, preferably 0; and x is from about 1.3 to about 10, preferablyfrom about 1.3 to about 3, most preferably from about 1.3 to about 2.7.The glycosyl is preferably derived from glucose. To prepare thesecompounds, the alcohol or alkylpolyethoxy alcohol is formed first andthen reacted with glucose, or a source of glucose, to form the glucoside(attachment at the 1-position). The additional glycosyl units can thenbe attached between their 1-position and the preceding glycosyl units2-, 3-, 4-, and/or 6-position, preferably predominantly the 2-position.

The condensation products of ethylene oxide with a hydrophobic baseformed by the condensation of propylene oxide with propylene glycol arealso suitable for use as the additional nonionic surfactant systems ofthe present invention. The hydrophobic portion of these compounds willpreferably have a molecular weight from about 1500 to about 1800 andwill exhibit water insolubility. The addition of polyoxyethylenemoieties to this hydrophobic portion tends to increase the watersolubility of the molecule as a whole, and the liquid character of theproduct is retained up to the point where the polyoxyethylene content isabout 50% of the total weight of the condensation product, whichcorresponds to condensation with up to about 40 moles of ethylene oxide.Examples of compounds of this type include certain of the commerciallyavailable Pluronic™ surfactants, marketed by BASF.

Also suitable for use as the nonionic surfactant of the nonionicsurfactant system of the present invention, are the condensationproducts of ethylene oxide with the product resulting from the reactionof propylene oxide and ethylenediamine. The hydrophobic moiety of theseproducts consists of the reaction product of ethylenediamine and excesspropylene oxide, and generally has a molecular weight of from about 2500to about 3000. This hydrophobic moiety is condensed with ethylene oxideto the extent that the condensation product contains from about 40% toabout 80% by weight of polyoxyethylene and has a molecular weight offrom about 5,000 to about 11,000. Examples of this type of nonionicsurfactant include certain of the commercially available Tetronic™compounds, marketed by BASF.

Preferred for use as the nonionic surfactant of the surfactant systemsof the present invention are polyethylene oxide condensates of alkylphenols, condensation products of primary and secondary aliphaticalcohols with from about 1 to about 25 moles of ethyleneoxide,alkylpolysaccharides, and mixtures hereof. Most preferred are C₈-C₁₄alkyl phenol ethoxylates having from 3 to 15 ethoxy groups and C₈-C₈alcohol ethoxylates (preferably C₁₀ avg.) having from 2 to 10 ethoxygroups, and mixtures thereof. Highly preferred nonionic surfactants arepolyhydroxy fatty acid amide surfactants of the formula:

wherein R¹ is H, or R¹ is C₁₋₄ hydrocarbyl, 2-hydroxyethyl,2-hydroxypropyl or a mixture thereof, R² is C₅₋₃₁ hydrocarbyl, and Z isa polyhydroxyhydrocarbyl having a linear hydrocarbyl chain with at least3 hydroxyls directly connected to the chain, or an alkoxylatedderivative thereof. Preferably, R¹ is methyl, R² is straight C₁₁₋₁₅alkyl or C₁₆₋₁₈ alkyl or alkenyl chain such as coconut alkyl or mixturesthereof, and Z is derived from a reducing sugar such as glucose,fructose, maltose or lactose, in a reductive amination reaction.

Highly preferred anionic surfactants include alkyl alkoxylated sulfatesurfactants. Examples hereof are water soluble salts or acids of theformula RO(A)_(m)SO3M wherein R is an unsubstituted C₁₀-C₂₄ alkyl orhydroxyalkyl group having a C₁₀-C₂₄ alkyl component, preferably aC₁₂-C₂₀ alkyl or hydroxyalkyl, more preferably C₁₂-C₁₈ alkyl orhydroxyalkyl, A is an ethoxy or propoxy unit, m is greater than zero,typically between about 0.5 and about 6, more preferably between about0.5 and about 3, and M is H or a cation which can be, for example, ametal cation (e.g., sodium, potassium, lithium, calcium, magnesium,etc.), ammonium or substituted-ammonium cation. Alkyl ethoxylatedsulfates as well as alkyl propoxylated sulfates are contemplated herein.Specific examples of substituted ammonium cations include methyl-,dimethyl, trimethyl-ammonium cations and quaternary ammonium cationssuch as tetramethyl-ammonium and dimethyl piperdinium cations and thosederived from alkylamines such as ethylamine, diethylamine,triethylamine, mixtures thereof, and the like. Exemplary surfactants areC₁₂-C₁₈ alkyl polyethoxylate (1.0) sulfate (C₁₂-C₁₈E(1.0)M), C₁₂-C₁₈alkyl polyethoxylate (2.25) sulfate (C₁₂-C₁₈(2.25)M, and C₁₂-C₁₈ alkylpolyethoxylate (3.0) sulfate (C₁₂-C₁₈E(3.0)M), and C₁₂-C₁₈ alkylpolyethoxylate (4.0) sulfate (C₁₂-C₁₈E(4.0)M), wherein M is convenientlyselected from sodium and potassium.

Suitable anionic surfactants to be used are alkyl ester sulfonatesurfactants including linear esters of C₈-C₂₀ carboxylic acids (i.e.,fatty acids) which are sulfonated with gaseous SO₃ according to “TheJournal of the American Oil Chemists Society”, 52 (1975), pp. 323-329.Suitable starting materials would include natural fatty substances asderived from tallow, palm oil, etc.

The preferred alkyl ester sulfonate surfactant, especially for laundryapplications, comprises alkyl ester sulfonate surfactants of thestructural formula:

wherein R³ is a C₈-C₂₀ hydrocarbyl, preferably an alkyl, or combinationthereof, R⁴ is a C₁-C₆ hydrocarbyl, preferably an alkyl, or combinationthereof, and M is a cation which forms a water soluble salt with thealkyl ester sulfonate. Suitable salt-forming cations include metals suchas sodium, potassium, and lithium, and substituted or unsubstitutedammonium cations, such as monoethanolamine, diethonolamine, andtriethanolamine. Preferably, R³ is C₁₀-C₁₆ alkyl, and R⁴ is methyl,ethyl or isopropyl. Especially preferred are the methyl ester sulfonateswherein R³ is C₁₀-C₁₆ alkyl.

Other suitable anionic surfactants include the alkyl sulfate surfactantswhich are water soluble salts or acids of the formula ROSO₃M wherein Rpreferably is a C₁₀-C₂₄ hydrocarbyl, preferably an alkyl or hydroxyalkylhaving a C₁₀-C₂₀ alkyl component, more preferably a C₁₂-C₁₈ alkyl orhydroxyalkyl, and M is H or a cation, e.g., an alkali metal cation (e.g.sodium, potassium, lithium), or ammonium or substituted ammonium (e.g.methyl-, dimethyl-, and trimethyl ammonium cations and quaternaryammonium cations such as tetramethyl-ammonium and dimethyl piperdiniumcations and quaternary ammonium cations derived from alkylamines such asethylamine, diethylamine, triethylamine, and mixtures thereof, and thelike). Typically, alkyl chains of C₁₂-C₁₆ are preferred for lower washtemperatures (e.g. below about 50 degrees Celsius) and C₁₆-C₁₈ alkylchains are preferred for higher wash temperatures (e.g. above about 50degrees Celsius).

Other anionic surfactants useful for detersive purposes can also beincluded in the laundry detergent compositions of the present invention.Theses can include salts (including, for example, sodium, potassium,ammonium, and substituted ammonium salts such as mono- di- andtriethanolamine salts) of soap, C₈-C₂₂ primary or secondaryalkanesulfonates, C₈-C₂₄ olefinsulfonates, sulfonated polycarboxylicacids prepared by sulfonation of the pyrolyzed product of alkaline earthmetal citrates, e.g., as described in British patent specification No.1,082,179, C₈-C₂₄ alkylpolyglycolethersulfates (containing up to 10moles of ethylene oxide); alkyl glycerol sulfonates, fatty acyl glycerolsulfonates, fatty oleyl glycerol sulfates, alkyl phenol ethylene oxideether sulfates, paraffin sulfonates, alkyl phosphates, isethionates suchas the acyl isethionates, N-acyl taurates, alkyl succinamates andsulfosuccinates, monoesters of sulfosuccinates (especially saturated andunsaturated C₁₂-C₁₈ monoesters) and diesters of sulfosuccinates(especially saturated and unsaturated C₆-C₁₂ diesters), acylsarcosinates, sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside (the nonionic nonsulfated compounds being describedbelow), branched primary alkyl sulfates, and alkyl polyethoxycarboxylates such as those of the formula RO(CH₂CH₂O)_(k)—CH₂C00-M+wherein R is a C₈-C₂₂ alkyl, k is an integer from 1 to 10, and M is asoluble salt forming cation. Resin acids and hydrogenated resin acidsare also suitable, such as rosin, hydrogenated rosin, and resin acidsand hydrogenated resin acids present in or derived from tall oil.

Alkylbenzene sulfonates are highly preferred. Especially preferred arelinear (straight-chain) alkyl benzene sulfonates (LAS) wherein the alkylgroup preferably contains from 10 to 18 carbon atoms.

Further examples are described in “Surface Active Agents and Detergents”(Vol. I and II by Schwartz, Perry and Berch). A variety of suchsurfactants are also generally disclosed in U.S. Pat. No. 3,929,678,(Column 23, line 58 through Column 29, line 23, herein incorporated byreference).

When included therein, the laundry detergent compositions of the presentinvention typically comprise from about 1% to about 40%, preferably fromabout 3% to about 20% by weight of such anionic surfactants.

The laundry detergent compositions of the present invention may alsocontain cationic, ampholytic, zwitterionic, and semi-polar surfactants,as well as the nonionic and/or anionic surfactants other than thosealready described herein.

Cationic detersive surfactants suitable for use in the laundry detergentcompositions of the present invention are those having one long-chainhydrocarbyl group. Examples of such cationic surfactants include theammonium surfactants such as alkyltrimethylammonium halogenides, andthose surfactants having the formula:[R²(OR³)_(y)][R⁴(OR³)_(y)]₂R⁵N+X−wherein R² is an alkyl or alkyl benzyl group having from about 8 toabout 18 carbon atoms in the alkyl chain, each R³ is selected form thegroup consisting of —CH₂CH₂—, —CH₂CH(CH₃)—, —CH₂CH(CH₂OH)—, —CH₂CH₂CH₂—,and mixtures thereof; each R⁴ is selected from the group consisting ofC₁-C₄ alkyl, C₁-C₄ hydroxyalkyl, benzyl ring structures formed byjoining the two R⁴ groups, —CH₂CHOHCHOHCOR⁶CHOHCH₂OH, wherein R⁶ is anyhexose or hexose polymer having a molecular weight less than about 1000,and hydrogen when y is not 0; R⁵ is the same as R⁴ or is an alkylchain,wherein the total number of carbon atoms or R² plus R⁵ is not morethan about 18; each y is from 0 to about 10,and the sum of the y valuesis from 0 to about 15; and X is any compatible anion.

Highly preferred cationic surfactants are the water soluble quaternaryammonium compounds useful in the present composition having the formula:R₁R₂R₃R₄N⁺X⁻  (i)wherein R₁ is C₈-C₁₈ alkyl, each of R₂, R₃ and R₄ is independently C₁-C₄alkyl, C₁-C₄ hydroxy alkyl, benzyl, and —(C₂H₄₀)_(x)H where x has avalue from 2 to 5, and X is an anion. Not more than one of R₂, R₃ or R₄should be benzyl.

The preferred alkyl chain length for R₁ is C₁₂-C₁₅, particularly wherethe alkyl group is a mixture of chain lengths derived from coconut orpalm kernel fat or is derived synthetically by olefin build up or OXOalcohols synthesis.

Preferred groups for R₂R₃ and R₄ are methyl and hydroxyethyl groups andthe anion X may be selected from halide, methosulphate, acetate andphosphate ions.

Examples of suitable quaternary ammonium compounds of formulae (i) foruse herein are:

-   coconut trimethyl ammonium chloride or bromide;-   coconut methyl dihydroxyethyl ammonium chloride or bromide;-   decyl triethyl ammonium chloride;-   decyl dimethyl hydroxyethyl ammonium chloride or bromide;-   C₁₂₋₁₅ dimethyl hydroxyethyl ammonium chloride or bromide;-   coconut dimethyl hydroxyethyl ammonium chloride or bromide;-   myristyl trimethyl ammonium methyl sulphate;-   lauryl dimethyl benzyl ammonium chloride or bromide;-   lauryl dimethyl (ethenoxy)₄ ammonium chloride or bromide;-   choline esters (compounds of formula (i) wherein R₁ is-   di-alkyl imidazolines [compounds of formula (i)].

Other cationic surfactants useful herein are also described in U.S. Pat.No. 4,228,044 and in EP0 000 224.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 25%, preferably fromabout 1% to about 8% by weight of such cationic surfactants.

Ampholytic surfactants are also suitable for use in the laundrydetergent compositions of the present invention. These surfactants canbe broadly described as aliphatic derivatives of secondary or tertiaryamines, or aliphatic derivatives of heterocyclic secondary and tertiaryamines in which the aliphatic radical can be straight- orbranched-chain. One of the aliphatic substituents contains at leastabout 8 carbon atoms, typically from about 8 to about 18 carbon atoms,and at least one contains an anionic water-solubilizing group, e.g.carboxy, sulfonate, sulfate. See U.S. Pat. No. 3,929,678 (column 19,lines 18-35) for examples of ampholytic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such ampholytic surfactants.

Zwitterionic surfactants are also suitable for use in laundry detergentcompositions. These surfactants can be broadly described as derivativesof secondary and tertiary amines, derivatives of heterocyclic secondaryand tertiary amines, or derivatives of quaternary ammonium, quaternaryphosphonium or tertiary sulfonium compounds. See U.S. Pat. No. 3,929,678(column 19, line 38 through column 22, line 48) for examples ofzwitterionic surfactants.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such zwitterionic surfactants.

Semi-polar nonionic surfactants are a special category of nonionicsurfactants which include water-soluble amine oxides containing onealkyl moiety of from about 10 to about 18 carbon atoms and 2 moietiesselected from the group consisting of alkyl groups and hydroxyalkylgroups containing from about 1 to about 3 carbon atoms; watersolublephosphine oxides containing one alkyl moiety of from about 10 to about18 carbon atoms and 2 moieties selected from the group consisting ofalkyl groups and hydroxyalkyl groups containing from about 1 to about 3carbon atoms; and water-soluble sulfoxides containing one alkyl moietyfrom about 10 to about 18 carbon atoms and a moiety selected from thegroup consisting of alkyl and hydroxyalkyl moieties of from about 1 toabout 3 carbon atoms.

Semi-polar nonionic detergent surfactants include the amine oxidesurfactants having the formula:

wherein R³ is an alkyl, hydroxyalkyl, or alkyl phenyl group or mixturesthereof containing from about 8 to about 22 carbon atoms; R⁴ is analkylene or hydroxyalkylene group containing from about 2 to about 3carbon atoms or mixtures thereof; x is from 0 to about 3: and each R⁵ isan alkyl or hydroxyalkyl group containing from about 1 to about 3 carbonatoms or a polyethylene oxide group containing from about 1 to about 3ethylene oxide groups. The R⁵ groups can be attached to each other,e.g., through an oxygen or nitrogen atom, to form a ring structure.

These amine oxide surfactants in particular include C₁₀-C₁₈ alkyldimethyl amine oxides and C₈-C₁₂ alkoxy ethyl dihydroxy ethyl amineoxides.

When included therein, the laundry detergent compositions of the presentinvention typically comprise from 0.2% to about 15%, preferably fromabout 1% to about 10% by weight of such semi-polar nonionic surfactants.

Builder System

The compositions according to the present invention may further comprisea builder system. Any conventional builder system is suitable for useherein including aluminosilicate materials, silicates, polycarboxylatesand fatty acids, materials such as ethylenediamine tetraacetate, metalion sequestrants such as aminopolyphosphonates, particularlyethylenediamine tetramethylene phosphonic acid and diethylene triaminepentamethylenephosphonic acid. Though less preferred for obviousenvironmental reasons, phosphate builders can also be used herein.

Suitable builders can be an inorganic ion exchange material, commonly aninorganic hydrated aluminosilicate material, more particularly ahydrated synthetic zeolite such as hydrated zeolite A, X, B, HS or MAP.

Another suitable inorganic builder material is layered silicate, e.g.SKS-6 (Hoechst). SKS-6 is a crystalline layered silicate consisting ofsodium silicate (Na₂Si₂O₅).

Suitable polycarboxylates containing one carboxy group include lacticacid, glycolic acid and ether derivatives thereof as disclosed inBelgian Patent Nos. 831,368, 821,369 and 821,370. Polycarboxylatescontaining two carboxy groups include the water-soluble salts ofsuccinic acid, malonic acid, (ethylenedioxy) diacetic acid, maleic acid,diglycollic acid, tartaric acid, tartronic acid and fumaric acid, aswell as the ether carboxylates described in German Offenle-enschrift2,446,686, and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinylcarboxylates described in Belgian Patent No. 840,623. Polycarboxylatescontaining three carboxy groups include, in particular, water-solublecitrates, aconitrates and citraconates as well as succinate derivativessuch as the carboxymethyloxysuccinates described in British Patent No.1,379,241, lactoxysuccinates described in Netherlands Application7205873, and the oxypolycarboxylate materials such as2-oxa-1,1,3-propane tricarboxylates described in British Patent No.1,387,447.

Polycarboxylates containing four carboxy groups include oxydisuccinatesdisclosed in British Patent No. 1,261,829, 1,1,2,2,-ethanetetracarboxylates, 1,1,3,3-propane tetracarboxylates containing sulfosubstituents include the sulfosuccinate derivatives disclosed in BritishPatent Nos. 1,398,421 and 1,398,422 and in U.S. Pat. No. 3,936,448, andthe sulfonated pyrolysed citrates described in British Patent No.1,082,179, while polycarboxylates containing phosphone substituents aredisclosed in British Patent No. 1,439,000.

Alicyclic and heterocyclic polycarboxylates includecyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienidepentacarboxylates,2,3,4,5-tetrahydro-furan-cis,cis,cis-tetracarboxylates,2,5-tetrahydro-furan-cis, discarboxylates,2,2,5,5,-tetrahydrofuran-tetracarboxylates,1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives ofpolyhydric alcohols such as sorbitol, mannitol and xylitol. Aromaticpolycarboxylates include mellitic acid, pyromellitic acid and thephthalic acid derivatives disclosed in British Patent No. 1,425,343.

Of the above, the preferred polycarboxylates are hydroxy-carboxylatescontaining up to three carboxy groups per molecule, more particularlycitrates.

Preferred builder systems for use in the present compositions include amixture of a water-insoluble aluminosilicate builder such as zeolite Aor of a layered silicate (SKS-6), and a water-soluble carboxylatechelating agent such as citric acid.

A suitable chelant for inclusion in the detergent composi-ions inaccordance with the invention is ethylenediamine-N,N′-disuccinic acid(EDDS) or the alkali metal, alkaline earth metal, ammonium, orsubstituted ammonium salts thereof, or mixtures thereof. Preferred EDDScompounds are the free acid form and the sodium or magnesium saltthereof. Examples of such preferred sodium salts of EDDS include Na₂EDDSand Na₄EDDS. Examples of such preferred magnesium salts of EDDS includeMgEDDS and Mg₂EDDS. The magnesium salts are the most preferred forinclusion in compositions in accordance with the invention.

Preferred builder systems include a mixture of a water-insolublealuminosilicate builder such as zeolite A, and a water solublecarboxylate chelating agent such as citric acid.

Other builder materials that can form part of the builder system for usein granular compositions include inorganic materials such as alkalimetal carbonates, bicarbonates, silicates, and organic materials such asthe organic phosphonates, amino polyalkylene phosphonates and aminopolycarboxylates.

Other suitable water-soluble organic salts are the homo- or co-polymericacids or their salts, in which the polycarboxylic acid comprises atleast two carboxyl radicals separated form each other by not more thantwo carbon atoms.

Polymers of this type are disclosed in GB-A-1,596,756. Examples of suchsalts are polyacrylates of MW 2000-5000 and their copolymers with maleicanhydride, such copolymers having a molecular weight of from 20,000 to70,000, especially about 40,000.

Detergency builder salts are normally included in amounts of from 5% to80% by weight of the composition. Preferred levels of builder for liquiddetergents are from 5% to 30%.

Enzymes

Preferred detergent compositions, in addition to the pectate lyasepreparation of the invention, comprise other enzyme(s) which providescleaning performance and/or fabric care benefits. In accordance with thepresent invention, additional enzymes may be modified in order toimprove the oxidation- and calcium-depletion stability.

Such enzymes include proteases, lipases, cutinases, amylases,cellulases, peroxidases, oxidases (e.g. laccases), hemicellulases, suchas mannanases, xylanases, galactanases, arabinofuranosidases, esterases,lichenases, arabinanases and other pectate lyases.

Proteases: Any protease suitable for use in alkaline solutions can beused. Suitable proteases include those of animal, vegetable or microbialorigin. Microbial origin is preferred. Chemically or geneticallymodified mutants are included. The protease may be a serine protease,preferably an alkaline microbial protease or a trypsin-like protease.Examples of alkaline proteases are subtilisins, especially those derivedfrom Bacillus, e.g., substilisin Novo, subtilisin Carlsberg, subtilisin309, substilisin 147 and substilisin 168 (described in WO 89/06279).Examples of trypsin-like proteases are trypsin (e.g. of porcine orbovine origin) and the Fusarium protease described in WO 89/06270.

Preferred commercially available protease enzymes include those soldunder the trade names Alcalase, Savinase, Primase, Durazym, and Esperaseby Novo Nordisk A/S (Denmark), those sold under the tradename Maxatase,Maxacal, Maxapem, Properase, Purafect and Purafect OXP by GenencorInternational, and those sold under the tradename Opticlean and Optimaseby Solvay Enzymes. Protease enzymes may be incorporated into thecompositions in accordance with the invention at a level of from0.00001% to 2% of enzyme protein by weight of the composition,preferably at a level of from 0.0001% to 1% of enzyme protein by weightof the composition, more preferably at a level of from 0.001% to 0.5% ofenzyme protein by weight of the composition, even more preferably at alevel of from 0.01 % to 0.2% of enzyme protein by weight of thecomposition.

Lipases: Any lipase suitable for use in alkaline solutions can be used.Suitable lipases include those of bacterial or fungal origin. Chemicallyor genetically modified mutants are included.

Examples of useful lipases include a Humicola lanuginosa lipase, e.g.,as described in EP 258 068 and EP 305 216, a Rhizomucor miehei lipase,e.g., as described in EP 238 023, a Candida lipase, such as a C.antarctica lipase, e.g., the C. antarctica lipase A or B described in EP214 761, a Pseudomonas lipase such as a P. alcaligenes and P .pseudoalcaligenes lipase, e.g., as described in EP 218 272, a P. cepacialipase, e.g., as described in EP 331 376, a P. stutzeri lipase, e.g., asdisclosed in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase,e.g., a B. subtilis lipase (Dartois et al., (1993), Biochemica etBiophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP64/744992) and a B. pumilus lipase (WO 91/16422).

Furthermore, a number of cloned lipases may be useful, including thePenicillium camembertii lipase described by Yamaguchi et al., (1991),Gene 103, 61-67), the Geotricum candidum lipase (Schimada, Y. et al.,(1989), J. Biochem., 106, 383-388), and various Rhizopus lipases such asa R. delemar lipase (Hass, M.J et al., (1991), Gene 109, 117-113), a R.niveus lipase (Kugimiya et al., (1992), Biosci. Biotech. Biochem. 56,716-719) and a R. oryzae lipase.

Other types of lipolytic enzymes such as cutinases may also be useful,e.g., a cutinase derived from Pseudomonas mendocina as described in WO88/09367, or a cutinase derived from Fusarium solani pisi (e.g.described in WO 90/09446).

Especially suitable lipases are lipases such as M1 Lipase™, Luma fast™and Lipomax™ (Genencor), Lipolase™ and Lipolase Ultra™ (Novo NordiskA/S), and Lipase P “Amano”(Amano Pharmaceutical Co. Ltd.).

The lipases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Amylases: Any amylase (alfa and/or beta) suitable for use in alkalinesolutions can be used. Suitable amylases include those of bacterial orfungal origin. Chemically or genetically modified mutants are included.Amylases include, for example, alfa-amylases obtained from a specialstrain of B. lichenifonnis, described in more detail in GB 1,296,839.Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (available from Novo Nordisk A/S) and Rapidase™, Maxamyl P™,Purastar™ and Purastar OxAm™ (available from Genencor). Further, anamylase with more than 70% homology to SP707 (Tsukamoto, A. et al, 1988.Biochem. Biophys. Res. Commun. 151:25) or K38 (Kao Corp. EP1022334) issuitable”.

The amylases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein byweight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Cellulases: Any cellulase suitable for use in alkaline solutions can beused. Suitable cellulases include those of bacterial or fungal origin.Chemically or genetically modified mutants are included. Suitablecellulases are disclosed in U.S. Pat. No. 4,435,307, which disclosesfungal cellulases produced from Humicola insolens. Especially suitablecellulases are the cellulases having colour care benefits. Examples ofsuch cellulases are cellulases described in European patent applicationNo. 0 495 257.

Commercially available cellulases include Celluzyme™ produced by astrain of Humicola insolens, (Novo Nordisk A/S), and KAC-500(B)™ (KaoCorporation).

Cellulases are normally incorporated in the detergent composition at alevel of from 0.00001% to 2% of enzyme protein by weight of thecomposition, preferably at a level of from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level of from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level of from 0.01% to 0.2% of enzyme protein by weightof the composition.

Peroxidases/Oxidases: Peroxidase enzymes are used in combination withhydrogen peroxide or a source thereof (e.g. a percarbonate, perborate orpersulfate). Oxidase enzymes are used in combination with oxygen. Bothtypes of enzymes are used for “soluton bleaching”, i.e. to preventtransfer of a textile dye from a dyed fabric to another fabric when saidfabrics are washed together in a wash liquor, preferably together withan enhancing agent as described in e.g. WO 94/12621 and WO 95/01426.Suitable peroxidases/oxidases include those of plant, bacterial orfungal origin. Chemically or genetically modified mutants are included.

Peroxidase and/or oxidase enzymes are normally incorporated in thedetergent composition at a level of from 0.00001% to 2% of enzymeprotein by weight of the composition, preferably at a level of from0.0001% to 1% of enzyme protein by weight of the composition, morepreferably at a level of from 0.001% to 0.5% of enzyme protein by weightof the composition, even more preferably at a level of from 0.01% to0.2% of enzyme protein by weight of the composition.

Pectate lyases: Pectate lyases have been cloned from different bacterialgenera such as Erwinia, Pseudomonas, Klebsiella and Xanthomonas. Alsofrom Bacillus subtilis (Nasser et al. (1993) FEBS 335:319-326) andBacillus sp. YA-14 (Kim et al. (1994) Biosci. Biotech. Biochem.58:947-949) cloning of a pectate lyase has been described.

The pectate lyases are generally characterised by an alkaline pH optimumand an absolute requirement for divalent cations, Ca²⁺ being the moststimulatory.

Mixtures of the above mentioned enzymes are encompassed herein, inparticular a mixture of a protease, an amylase, a lipase and/or acellulase.

The pectate lyase of the invention, or any other enzyme incorporated inthe detergent composition, is normally incorporated in the detergentcomposition at a level from 0.00001% to 2% of enzyme protein by weightof the composition, preferably at a level from 0.0001% to 1% of enzymeprotein by weight of the composition, more preferably at a level from0.001% to 0.5% of enzyme protein by weight of the composition, even morepreferably at a level from 0.01% to 0.2% of enzyme protein by weight ofthe composition.

Bleaching agents: Additional optional detergent ingredients that can beincluded in the detergent compositions of the present invention includebleaching agents such as PB1, PB4 and percarbonate with a particle sizeof 400-800 microns. These bleaching agent components can include one ormore oxygen bleaching agents and, depending upon the bleaching agentchosen, one or more bleach activators. When present oxygen bleachingcompounds will typically be present at levels of from about 1% to about25%. In general, bleaching compounds are optional added components innon-liquid formulations, e.g. granular detergents.

The bleaching agent component for use herein can be any of the bleachingagents useful for detergent compositions including oxygen bleaches aswell as others known in the art.

The bleaching agent suitable for the present invention can be anactivated or non-activated bleaching agent.

One category of oxygen bleaching agent that can be used encompassespercarboxylic acid bleaching agents and salts thereof. Suitable examplesof this class of agents include magnesium monoperoxyphthalatehexahydrate, the magnesium salt of meta-chloro perbenzoic acid,4-nonylamino-4-oxoperoxybutyric acid and diperoxydodecanedioic acid.Such bleaching agents are disclosed in U.S. Pat. No. 4,483,781, U.S.Pat. No. 740,446, EP 0 133 354 and U.S. Pat. No. 4,412,934. Highlypreferred bleaching agents also include 6-nonylamino-6-oxoperoxycaproicacid as described in U.S. Pat. No. 4,634,551.

Another category of bleaching agents that can be used encompasses thehalogen bleaching agents. Examples of hypohalite bleaching agents, forexample, include trichloro isocyanuric acid and the sodium and potassiumdichloroisocyanurates and N-chloro and N-bromo alkane sulphonamides.Such materials are normally added at 0.5-10% by weight of the finishedproduct, preferably 1-5% by weight.

The hydrogen peroxide releasing agents can be used in combination withbleach activators such as tetra-acetylethylenediamine (TAED),nonanoyloxybenzenesulfonate (NOBS, described in U.S. Pat. No.4,412,934), 3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS,described in EP 120 591) or pentaacetylglucose (PAG), which areperhydrolyzed to form a peracid as the active bleaching species, leadingto improved bleaching effect. In addition, very suitable are the bleachactivators C8(6-octanamido-caproyl) oxybenzene-sulfonate,C9(6-nonanamido caproyl) oxybenzenesulfonate and C10 (6-decanamidocaproyl) oxybenzenesulfonate or mixtures thereof. Also suitableactivators are acylated citrate esters such as disclosed in EuropeanPatent Application No. 91870207.7.

Useful bleaching agents, including peroxyacids and bleaching systemscomprising bleach activators and peroxygen bleaching compounds for usein cleaning compositions according to the invention are described inapplication U.S. Ser. No. 08/136,626.

The hydrogen peroxide may also be present by adding an enzymatic system(i.e. an enzyme and a substrate therefore) which is capable ofgeneration of hydrogen peroxide at the beginning or during the washingand/or rinsing process. Such enzymatic systems are disclosed in EuropeanPatent Application EP 0 537 381.

Bleaching agents other than oxygen bleaching agents are also known inthe art and can be utilized herein. One type of non-oxygen bleachingagent of particular interest includes photoactivated bleaching agentssuch as the sulfonated zinc and/or aluminium phthalocyanines. Thesematerials can be deposited upon the substrate during the washingprocess. Upon irradiation with light, in the presence of oxygen, such asby hanging clothes out to dry in the daylight, the sulfonated zincphthalocyanine is activated and, consequently, the substrate isbleached. Preferred zinc phthalocyanine and a photoactivated bleachingprocess are described in U.S. Pat. No. 4,033,718. Typically, detergentcomposition will contain about 0.025% to about 1.25%, by weight, ofsulfonated zinc phthalocyanine.

Bleaching agents may also comprise a manganese catalyst. The manganesecatalyst may, e.g., be one of the compounds described in “Efficientmanganese catalysts for low-temperature bleaching”, Nature 369, 1994,pp. 637-639.

Suds suppressors: Another optional ingredient is a suds suppressor,exemplified by silicones, and silica-silicone mixtures. Silicones cangenerally be represented by alkylated polysiloxane materials, whilesilica is normally used in finely divided forms exemplified by silicaaerogels and xerogels and hydrophobic silicas of various types. Thesesmaterials can be incorporated as particulates, in which the sudssuppressor is advantageously releasably incorporated in a water-solubleor waterdispersible, substantially non surface-active detergentimpermeable carrier. Alternatively the suds suppressor can be dissolvedor dispersed in a liquid carrier and applied by spraying on to one ormore of the other components.

A preferred silicone suds controlling agent is disclosed in U.S. Pat.No. 3,933,672. Other particularly useful suds suppressors are theself-emulsifying silicone suds suppressors, described in German PatentApplication DTOS 2,646,126. An example of such a compound is DC-544,commercially available form Dow Corning, which is a siloxane-glycolcopolymer. Especially preferred suds controlling agent are the sudssuppressor system comprising a mixture of silicone oils and2-alkyl-alkanols. Suitable 2-alkyl-alkanols are 2-butyl-octanols whichare commercially available under the trade name Isofol 12 R.

Such suds suppressor system are described in European Patent ApplicationEP 0 593 841.

Especially preferred silicone suds controlling agents are described inEuropean Patent Application No. 92201649.8. Said compositions cancomprise a silicone/silica mixture in combination with fumed nonporoussilica such as Aerosil^(R).

The suds suppressors described above are normally employed at levels offrom 0.001% to 2% by weight of the composition, preferably from 0.01% to1% by weight.

Other components: Other components used in detergent compositions may beemployed such as soil-suspending agents, soil-releasing agents, opticalbrighteners, abrasives, bactericides, tarnish inhibitors, coloringagents, and/or encapsulated or nonencapsulated perfumes.

Especially suitable encapsulating materials are water soluble capsuleswhich consist of a matrix of polysaccharide and polyhydroxy compoundssuch as described in GB 1,464,616.

Other suitable water soluble encapsulating materials comprise dextrinsderived from ungelatinized starch acid esters of substituteddicarboxylic acids such as described in U.S. Pat. No. 3,455,838. Theseacid-ester dextrins are, preferably, prepared from such starches as waxymaize, waxy sorghum, sago, tapioca and potato. Suitable examples of saidencapsulation materials include N-Lok manufactured by National Starch.The N-Lok encapsulating material consists of a modified maize starch andglucose. The starch is modified by adding monofunctional substitutedgroups such as octenyl succinic acid anhydride.

Antiredeposition and soil suspension agents suitable herein includecellulose derivatives such as methylcellulose, carboxymethylcelluloseand hydroxyethylcellulose, and homo- or co-polymeric polycarboxylicacids or their salts. Polymers of this type include the polyacrylatesand maleic anhydride-acrylic acid copolymers previously mentioned asbuilders, as well as copolymers of maleic anhydride with ethylene,methylvinyl ether or methacrylic acid, the maleic anhydride constitutingat least 20 mole percent of the copolymer. These materials are normallyused at levels of from 0.5% to 10% by weight, more preferably form 0.75%to 8%, most preferably from 1% to 6% by weight of the composition.

Preferred optical brighteners are anionic in character, examples ofwhich are disodium 4,4′-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2′disulphonate,disodium4,-4′-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2′-disulphonate,disodium4,4′-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2′-disulphonate,monosodium4′,4″-bis-(2,4-dianilino-s-tri-azin6ylamino)stilbene-2-sulphonate,disodium4,4′-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamino)stilbene-2,2′-disulphonate,di-sodium4,4′-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2′disulphonate,di-so-dium 4,4′bis(2-anilino4-(1-methyl-2-hydroxyethylamino)-s-triazin-6-ylami-no)stilbene-2,2′disulphonate,sodium 2(stilbyl-4″-(naphtho-1′,2′:4,5)-1,2,3,-triazole-2″-sulphonateand 4,4′-bis(2-sulphostyryl)biphenyl.

Other useful polymeric materials are the polyethylene glycols,particularly those of molecular weight 1000-10000, more particularly2000 to 8000 and most preferably about 4000. These are used at levels offrom 0.20% to 5% more preferably from 0.25% to 2.5% by weight. Thesepolymers and the previously mentioned homo- or co-polymericpolycarboxylate salts are valuable for improving whiteness maintenance,fabric ash deposition, and cleaning performance on clay, proteinaceousand oxidizable soils in the presence of transition metal impurities.

Soil release agents useful in compositions of the present invention areconventionally copolymers or terpolymers of terephthalic acid withethylene glycol and/or propylene glycol units in various arrangements.Examples of such polymers are disclosed in U.S. Pat. Nos. 4,116,885 and4,711,730 and EP 0 272 033. A particular preferred polymer in accordancewith EP 0 272 033 has the formula:(CH₃(PEG)₄₃)_(0.75)(POH)_(0.25) [T-PO)_(2.8)(T-PEG)_(0.4)]T(POH)_(0.25)((PEG)₄₃CH₃)_(0.75)

where PEG is —(OC₂H₄)0-, PO is (OC₃H₆O) and T is (pOOC₆H₄CO).

Also very useful are modified polyesters as random copolymers ofdimethyl terephthalate, dimethyl sulfoisophthalate, ethylene glycol and1,2-propanediol, the end groups consisting primarily of sulphobenzoateand secondarily of mono esters of ethylene glycol and/or1,2-propanediol. The target is to obtain a polymer capped at both end bysulphobenzoate groups, “primarily”, in the present context most of saidcopolymers herein will be endcapped by sulphobenzoate groups. However,some copolymers will be less than fully capped, and therefore their endgroups may consist of monoester of ethylene glycol and/or1,2-propanediol, thereof consist “secondarily” of such species.

The selected polyesters herein contain about 46% by weight of dimethylterephthalic acid, about 16% by weight of 1,2-propanediol, about 10% byweight ethylene glycol, about 13% by weight of dimethyl sulfobenzoicacid and about 15% by weight of sulfoisophthalic acid, and have amolecular weight of about 3.000. The polyesters and their method ofpreparation are described in detail in EP 311 342.

Softening agents: Fabric softening agents can also be incorporated intolaundry detergent compositions in accordance with the present invention.These agents may be inorganic or organic in type. Inorganic softeningagents are exemplified by the smectite clays disclosed in GB-A-1 400898and in U.S. Pat. No. 5,019,292. Organic fabric softening agents includethe water insoluble tertiary amines as disclosed in GB-A1 514 276 and EP0 011 340 and their combination with mono C₁₂-C₁₄ quaternary ammoniumsalts are disclosed in EP-B-0 026 528 and di-long-chain amides asdisclosed in EP 0 242 919. Other useful organic ingredients of fabricsoftening systems include high molecular weight polyethylene oxidematerials as disclosed in EP 0 299 575 and 0 313 146.

Levels of smectite clay are normally in the range from 5% to 15%, morepreferably from 8% to 12% by weight, with the material being added as adry mixed component to the remainder of the formulation. Organic fabricsoftening agents such as the water-insoluble tertiary amines or dilongchain amide materials are incorporated at levels of from 0.5% to 5% byweight, normally from 1% to 3% by weight whilst the high molecularweight polyethylene oxide materials and the water soluble cationicmaterials are added at levels of from 0.1% to 2%, normally from 0.15% to1.5% by weight. These materials are normally added to the spray driedportion of the composition, although in some instances it may be moreconvenient to add them as a dry mixed particulate, or spray them asmolten liquid on to other solid components of the composition.

Polvmeric dye-transfer inhibiting agents: The detergent compositionsaccording to the present invention may also comprise from 0.001% to 10%,preferably from 0.01% to 2%, more preferably form 0.05% to 1% by weightof polymeric dye-transfer inhibiting agents. Said polymeric dye-transferinhibiting agents are normally incorporated into detergent compositionsin order to inhibit the transfer of dyes from colored fabrics ontofabrics washed therewith. These polymers have the ability of complexingor adsorbing the fugitive dyes washed out of dyed fabrics before thedyes have the opportunity to become attached to other articles in thewash.

Especially suitable polymeric dye-transfer inhibiting agents arepolyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone andN-vinylimidazole, polyvinylpyrrolidone polymers, polyvinyloxazolidonesand polyvinylimidazoles or mixtures thereof.

Addition of such polymers also enhances the performance of the enzymesaccording the invention.

The detergent composition according to the invention can be in liquid,paste, gels, bars or granular forms.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) and mayoptionally be coated by methods known in the art. Examples of waxycoating materials are poly(ethylene oxide) products(polyethylene-glycol, PEG) with mean molecular weights of 1000 to 20000;ethoxylated nonylphenols having from 16 to 50 ethylene oxide units;ethoxylated fatty alcohols in which the alcohol contains from 12 to 20carbon atoms and in which there are 15 to 80 ethylene oxide units; fattyalcohols; fatty acids; and mono- and di- and triglycerides of fattyacids. Examples of film-forming coating materials suitable forapplication by fluid bed techniques are given in GB 1483591.

Granular compositions according to the present invention can also be in“compact form”, i.e. they may have a relatively higher density thanconventional granular detergents, i.e. form 550 to 950 g/l; in suchcase, the granular detergent compositions according to the presentinvention will contain a lower amount of “Inorganic filler salt”,compared to conventional granular detergents; typical filler salts arealkaline earth metal salts of sulphates and chlorides, typically sodiumsulphate; “Compact” detergent typically comprise not more than 10%filler salt. The liquid compositions according to the present inventioncan also be in “concentrated form”, in such case, the liquid detergentcompositions according to the present invention will contain a loweramount of water, compared to conventional liquid detergents. Typically,the water content of the concentrated liquid detergent is less than 30%,more preferably less than 20%, most preferably less than 10% by weightof the detergent compositions.

The compositions of the invention may for example, be formulated as handand machine laundry detergent compositions including laundry additivecompositions and compositions suitable for use in the pretreatment ofstained fabrics, rinse added fabric softener compositions, andcompositions for use in general household hard surface cleaningoperations and dishwashing operations.

The following examples are meant to exemplify compositions for thepresent invention, but are not necessarily meant to limit or otherwisedefine the scope of the invention.

In the detergent compositions, the abbreviated component identificationshave the following meanings:

-   -   LAS: Sodium linear C₁₂ alkyl benzene sulfonate    -   TAS: Sodium tallow alkyl sulfate    -   XYAS: Sodium C_(1X)-C_(1Y) alkyl sulfate    -   SS: Secondary soap surfactant of formula 2-butyl octanoic acid    -   25EY: A C₁₂-C₁₅ predominantly linear primary alcohol condensed        with an average of Y moles of ethylene oxide    -   45EY: A C₁₄-C₁₅ predominantly linear primary alcohol condensed        with an average of Y moles of ethylene oxide    -   XYEZS: C_(1X)-C_(1Y) sodium alkyl sulfate condensed with an        average of Z moles of ethylene oxide per mole

Nonionic: C₁₃-C₁₅ mixed ethoxylated/propoxylated fatty alcohol with anaverage degree of ethoxylation of 3.8 and an average degree ofpropoxylation of 4.5 sold under the tradename Plurafax LF404 by BASFGmbh

-   -   CFAA: C₁₂-C₁₄ alkyl N-methyl glucamide    -   TFAA: C₁₆-C₁₈ alkyl N-methyl glucamide    -   Silicate: Amorphous Sodium Silicate (SiO₂:Na₂O ratio=2.0)    -   NaSKS-6: Crystalline layered silicate of formula δ—Na₂Si₂O₅    -   Carbonate: Anhydrous sodium carbonate    -   Phosphate: Sodium tripolyphosphate    -   MAIM: Copolymer of 1:4 maleic/acrylic acid, average molecular        weight about 80,000    -   Polyacrylate: Polyacrylate homopolymer with an average molecular        weight of 8,000 sold under the tradename PA30 by BASF Gmbh    -   Zeolite A: Hydrated Sodium Aluminosilicate of formula        Na₁₂(AlO₂SiO₂)₁₂. 27H₂O having a primary particle size in the        range from 1 to 10 micrometers    -   Citrate: Tri-sodium citrate dihydrate    -   Citric: Citric Acid    -   Perborate: Anhydrous sodium perborate monohydrate bleach,        empirical formula NaBO₂.H₂O₂    -   PB4: Anhydrous sodium perborate tetrahydrate    -   Percarbonate: Anhydrous sodium percarbonate bleach of empirical        formula 2Na₂CO₃.3H₂O₂    -   TAED: Tetraacetyl ethylene diamine    -   CMC: Sodium carboxymethyl cellulose    -   DETPMP: Diethylene triamine penta (methylene phosphonic acid),        marketed by Monsanto under the Tradename Dequest 2060    -   PVP: Polyvinylpyrrolidone polymer    -   EDDS: Ethylenediamine-N,N′-disuccinic acid, [S,S] isomer in the        form of the sodium salt    -   Suds 25% paraffin wax Mpt 50° C., 17% hydrophobic silica, 58%        Suppressor: paraffin oil    -   Granular Suds 12% Silicone/silica, 18% stearyl alcohol, 70%        suppressor: starch in granular form    -   Sulphate: Anhydrous sodium sulphate    -   HMWPEO: High molecular weight polyethylene oxide    -   TAE 25: Tallow alcohol ethoxylate (25)

Detergent Example I

A granular fabric cleaning composition in accordance with the inventionmay be prepared as follows: Sodium linear C₁₂ alkyl 6.5 benzenesulfonate Sodium sulfate 15.0 Zeolite A 26.0 Sodium nitrilotriacetate5.0 Enzyme of the invention 0.1 PVP 0.5 TAED 3.0 Boric acid 4.0Perborate 18.0 Phenol sulphonate 0.1 Minors Up to 100

Detergent Example II

A compact granular fabric cleaning composition (density 800 g/l) inaccord with the invention may be prepared as follows: 45AS 8.0 25E3S 2.025E5 3.0 25E3 3.0 TFAA 2.5 Zeolite A 17.0 NaSKS- 12.0 Citric acid 3.0Carbonate 7.0 MA/AA 5.0 CMC 0.4 Enzyme of the invention 0.1 TAED 6.0Percarbonate 22.0 EDDS 0.3 Granular suds suppressor 3.5 water/minors Upto 100%

Detergent Example III

Granular fabric cleaning compositions in accordance with the inventionwhich are especially useful in the laundering of coloured fabrics wereprepared as follows: LAS 10.7 — TAS 2.4 — TFAA — 4.0 45AS 3.1 10.0 45E74.0 — 25E3S — 3.0 68E11 1.8 — 25E5 — 8.0 Citrate 15.0 7.0 Carbonate — 10Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 — 9.0 MA/AA 5.0 5.0DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05 Silicate 2.5 — Sulphate5.2 3.0 PVP 0.5 — Poly (4-vinylpyridine)-N- — 0.2 Oxide/copolymer ofvinyl- imidazole and vinyl- pyrrolidone Perborate 1.0 — Phenol sulfonate0.2 — Water/Minors Up to 100%

Detergent Example IV

Granular fabric cleaning compositions in accordance with the inventionwhich provide “Softening through the wash” capability may be prepared asfollows: 45AS — 10.0 LAS 7.6 — 68AS 1.3 — 45E7 4.0 — 25E3 — 5.0Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride Citrate 5.03.0 Na-SKS-6 — 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0 DETPMP 0.4 0.4Perborate 15.0 — Percarbonate — 15.0 TAED 5.0 5.0 Smectite clay 10.010.0 HMWPEO — 0.1 Enzyme of the invention 0.10 0.05 Silicate 3.0 5.0Carbonate 10.0 10.0 Granular suds suppressor 1.0 4.0 CMC 0.2 0.1Water/Minors Up to 100%

Detergent Example V

Heavy duty liquid fabric cleaning compositions in accordance with theinvention may be prepared as follows: A B LAS acid form — 25.0 Citricacid 5.0 2.0 25AS acid form 8.0 — 25AE2S acid form 3.0 — 25AE7 8.0 —CFAA 5 — DETPMP 1.0 1.0 Fatty acid 8 — Oleic acid — 1.0 Ethanol 4.0 6.0Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coco-alkyldimethyl- — 3.0 hydroxy ethyl ammonium chloride Smectite clay — 5.0 PVP2.0 — Water/Minors Up to 100%

Example 1: Measuring the Stability of Pectate Lyase in Liquid Detergent

The detergent stability of the pectate lyase variants of the presentinvention is assessed by measuring the activity of the variants afterincubation of an enzyme-detergent mixture, which is described below.

Residual Activity Assay

30 micro litre of an enzyme solution (culture supernatant or purifiedenzyme) is mixed with 1 ml of typical European or US heavy duty liquiddetergent in two sample tubes. One of the tubes is stored on ice whilethe other is incubated at 40 degrees Celsius for 90 minutes. Asreference 30 microlitre water is mixed with 1 ml of detergent andincubated on ice. After incubation, 9 ml of ice-cold water is added tothe samples, which are mixed vigorously and stored on ice until furtheranalysis.

The enzymatic activity is measured by first mixing 50 micro litre ofenzyme-detergent mixture with 5 ml of assay buffer (100 mM Tris-HCL,0.68 mM CaCl₂, pH 8.0), secondly from which solution, 75 micro litre ismixed with 75 micro litre freshly prepared substrate solution (1%polygalactoronic acid in assay buffer) and incubated at 40 degreesCelsius for 10 minutes. Thirdly, 100 micro litre of the incubationmixture is added into 100 micro litre stop-buffer (50 mM H₃PO₄) in aUV-transparent microtiterplate and the absorbance at 235 nm is measuredin a spectofotometer. The water-detergent sample is used to zero thespectofotometer.

Now the residual activity is calculated as the activity (A235absorbance) in the sample incubated at 40 degrees Celsius for 90 minutesrelative to the activity in the sample stored on ice:Residual activity (RA)=Absorbance[sample incubated at 40 degreesCelsius]/Absorbance[sample incubated at 0 degrees Celsius]×100

Thus the residual activity is equivalent to the detergent stability ofthe enzyme. Table 2 lists the improved residual activity of a number ofsubstitutions of the pectate lyase of SEQ ID NO:2 incubated in a typicalEuropean heavy duty liquid detergent. The majority of the substitutionsgive rise to more than 50 % improvement of the detergent stabilitycompared to the parent pectate lyase as shown in SEQ ID NO:2.Equivalently the substitutions of the pectate lyase of SEQ ID NO:2listed in Table 3 significantly improve the stability of the enzyme whenincubated in a typical US heavy duty liquid detergent. TABLE 2Substitutions in the pectate lyase of SEQ ID NO: 2 giving rise toincreased residual activity after incubation in European heavy dutyliquid detergent. Residual activity in Residual % of parent Mutationsactivity % enzyme Pectate lyase of SEQ ID NO: 2 (parent enzyme) 23 100Q40E 28 122 F251I 30 130.4 A332P 31 134.8 L106Q 31 134.8 R272H 31 134.8R272Y 31 134.8 A91E 33 143.5 K115I + K213E 33 143.5 K139I + K213N 33143.5 H5R + K257N + S302A 34 147.8 K139M 34 147.8 K87A 34 147.8 D48P 35152.2 K99I + I196V 35 152.2 T105P 35 152.2 K115A + K118A 36 156.5K115A + K118A + M122N 36 156.5 K115Q 36 156.5 K213T 36 156.5 V141E +C199S + K213E 36 156.5 K115I + Q146H 37 160.9 K257N 37 160.9 K7IE +K118E 38 165.2 S331P 38 165.2 T49P + N156S 38 165.2 K314N + S340P 39169.6 V141E + I235V 39 169.6 G46D + K257N 40 173.9 Q146H 40 173.9 K218P41 178 A305P 41 178 S28T + S30F + K334E + N363S 41 178 H193Y + S256C +V389I + A393V 42 183 R272C 42 183 D48E + L106Q + I140V + F215Y + K218E42 183 K213N + T258I 43 187 E9G + H31N + N50D + L106Q + A111E + T136S +V141L + F201L + N202K + 43 187 F215Y + G286A + A381D + H384N E9G +H31N + L106Q + D303S + A305P + T335S + H384N + S391N 44 191 E9G + H31N +D48E + L106Q + A111E + S301Y + D303S + A305P + T378S + H384N + S391N 45196 L45V + N50Y + N185H 46 200 N11Y + K87E + K99N 46 200 K115I + K213E48 209 E9G + D48E + L106Q + S316F + A381D 48 209 S30P + K115I + K139I +Q146H + S337C 49 213 K213N + T258I 50 217 K47R + K257N 50 217 E9G +H31N + D48E + L106Q + I140V + F215Y + D303S + A305P + T378S + H384N +S391N 51 222 H31N + T105A + L106Q + A111E + V141L + K218E + D303S +A305P + D326N + T335S + H384N + 51 222 S391N E9G + L106Q + I140V +G204R + F215Y + D303S + A305P + T378S + H384N + S391N 52 226 K26Q +K47N + L106Q + I140V + F215Y + D303S + A305P + T378S + H384N + S391N 53230 D48E + L106Q + I140V + F215Y + D303S + A305P + T378S + H384N + S391N56 243 D48E + L106Q + K213T + F215Y + K218L + A305P 57 248 L106Q + S337C57 248 K115I + K213N 57 248 E9G + H31N + T61A + G74D + L75A + N76D +K79A + D86N + E107K + T136S + V141L + 57 248 F215Y + K218E + V324A +A333E + S340K + G341S + T378S + H384N + S391N T52M + K139I + Q146H +I196V + K213N + K257N + T258I + N323M + S366N 57 248 N51Y + N186H +K213N + K257N 59 257 K213N 60 261 K139I + Q146H + K213N + T258I + S337C60 261 E9G + D48E + G74D + L75A + N76D + K79A + D86N + L106Q + T136S +V141L + 61 265 K148E + I194V + G204R + F215Y + D303S + A305P + T378S +H384N + S391N H31N + D48E + L106Q + T136S + V141L + I194V + S331T +H384N + K386R + S387A + I390T 62 270 K213T + K218L + A305P 64 278 S337C65 283 M122Q + K115Q + K118Q + K139I + Q146H + S337C 66 287 M64F +K213T + K218P + A305P 66 287 K213T + K218P + A305P + D48P + T105P +K139I + T258I 67 291 L45V + K139I + Q146H + N185H + N196V + S337C +G377E + S387I 67 291 M64F + L106Q + K213T + F215Y + K218L + A305P 68 296M64F + K213T + K218L + A305P 70 304 M64F + M122K + K118E + K213T +K218L + A305P 71 309 K213T + K218P + A305P + D48P + T105P + K139T 71 309D48E + M64F + L106Q + M122K + K118E + K213T + F215Y + K218L + A305P 72313 L45V + K139N + Q146H + I196V + T258I 72 313 K139I + V141G + Q146H +S337C + I357V 72 313 K99I + K139I + Q146H + N185H + I196V + K213N +K257N + T258I 73 317 K139I + Q146H + S337C 74 322 M64F + M122K + K118E +Q146H + K213T + K218L + A305P + S340Q 74 322 M122K + K118E + K139I +Q146H + S337C 75 326 K139N + E158N + Q146H + S337C 75 326 M64F + K118E +M122K + Q146H + K213N + K218P + A305P 75 326 K139I; Q146H + K257N +S337C 75 326 K99I + I196V + K213N 75 326 K139I + Q146H + R272C 75 326K139I + Q146H + N323M + S337C 75.5 328 M64F + M122K + K118E + Q146H +'I196V + K218L + A305P 76 330 L45V + N116D + N185H + K257N + T258I +S337C + Q356H 76 330 M64F + M237I + K139I + Q146H + S337C 77 335 D48P +M64F + K213T + K218L + A305P + S331P 77 335 M64F + K139N + Q146H +E158N + S337C 77 335 K139I + Q146H + S337C 77 335 M64F + M122E + K139I +Q146H + S337C 78 339 M64F + M122K + K118E + K139I + Q146H + S337C 78 339D48P + T105P + K139N + K213N + K218P + T258N + A305P + S331P 78 339M64F + K139S + Q146H + S337C 78 339 M64F + M122K + K118E + Q146H +K213T + K218L + A305P 79 343 K139I + Q146H + K213N + S331P + S337C 80348 Q146H + V338E 81 352 K139I + Q146H + N189D + S337R 81 352 M64F +K139H + Q146H + S337C 81 352 K139I + S337C + S340N 81.5 354 M64F +L106Q + K139I + Q146H + K213T + K218L + A305P + S337C 82 357 N76D +K139I + Q146I + S337C 82 357 M64F + K139F + Q146V + S337C 82 357 M64F +K139I + Q146H + Y308S + T335R + I336S + S337L + V338Y + F339I + S340A 82357 D48P + M64F + T105P + K139N + Q146H + K213N + K218P + T258N +A305P + S331P 83 361 M64F + V123I + K139I + Q146H + S337C 84 365 M64F +M122K + K118E + Q146H + K213T + K218L + A305P + S337C 84 365 K139I +Q146H + S256C 84 365 N37D + K139I + Q146H + K213E + S307R + S337C 84 365K139I + Q146H + S229T + S337C 84 365 M64F + K139S + Q146V + S337C 84 365M64F + M122V + K139Q + Q146L + S337C + K397D 84 365 M64F + K139I +Q146V + S337C 84 365 D48P + M64F + K118E + M122K + Q146H + D170N +K213N + K218P + A305P + S331P 85 370 L45V + K213N + K257N + S337C +T378G + H384N + S391N 85 370 M64F + M122K + K118E + Q146H + K213T +K218L + A305P + S340P 86 374 K139I + V141G + Q146H + V277D 86 374 M64F +K139L + Q146F + S337C 86 374 M64F + K99I + T105P + M122K + K118E +Q146H + K213N + K218P + A305P + S331P 86.5 376 K213T + K218P + A305P +D48P + T105P + K139I + T258I + S331P 87 378 D48P + M64F + T105P +K139N + Q146H + K213T + K218P + T258I + A305P + S331P 89 387 M64F +K139I + Q146H + S337C 90 391 D48P + M64F + T105P + K139I + Q146H +K213T + K218P + T258I + A305P + S331P 90.5 393 M64F + K139I + Q146H +K213T + K218L + A305P + S337C 91 396 D48P + T105P + K139N + K213T +K218P + T258I + A305P + S331P 91 396 M64F + M122K + K118E + Q146H +K213T + K218L + A305P + S337K 91 396 D48P + M64F + T105P + K139I +Q146H + K213T + K218P + T258I + A305P + S331P 91 396 V141E + C199S +K213E + Y295H 91 396 D48P + M64F + T105P + M122K + K118E + K213T +K218P + A305P + S331P 92 400 D48P + M64F + K139I + Q146H + K213T +K218L + A305P + S337K 92 400 D48P + T105P + K139N + K213T + K218P +T258I + A305P + S331P + S337R 92 400 D48P + M64F + T105P + K139I +Q146H + K213T + K218P + T258I + A305P + S331P + S337K 92 400 D48P +M4F + L106Q + K139I + Q146H + K213T + K218L + A305P + S337C 93 404D48P + M64F + T105P + K139I + Q146H + K148E + K213T + K218P + T258I +A305P + S331P + S337R 93 404 M64F + L75P + K139E + Q146V + G224S +S337C + G349R + V389I 93 404 M64F + K139I + Q146H + K213T + K218L +A305P + T258P + S331P + S337C 94 409 D48P + M64F + L106Q + K139I +Q146H + K213T + K218L + Y234H + A305P + S331P + S337C 94 409 M64F +M122K + K118E + Q146H + K213T + K218L + A305P + S337R 97 422 D48P +T105P + K139N + K213T + K218P + T258I + A305P + S331P + S337K 97 422D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I + A305P +S331P + S340P 97 422 M64F + K139I + Q146H + K213T + K218L + A305P +S331P + S337C 98 426 D48P + M64F + K139I + Q146H + K213T + K218L +A305P + S337C 98 426 D48P + M64F + K139I + Q146H + K213T + K218L +A305P + S337R 98 426 D48P + M64F + T105P + K139I + Q146H + K213T +K218P + T258I + A305P + S331P + S337R 99 430 D48P + M64F + T105P +K139I + Q146H + N189D + K213T + K218P + T258I + S298N + A305P + 100 435S331P + S337R D48P + M64F + T105P + K139I + Q146H + K213T + K218P +T258I + A305P + S331P + S337K + S340P 100 435 D48P + M64F + T105P +K139I + Q146H + K213T + K218P + T258I + A305P + S331P + K334E + 100 435S337K + S340P

TABLE 3 Substitutions in the pectate lyase of SEQ ID NO: 2 giving riseto increased residual activity after incubation in US heavy duty liquiddetergent. Residual activity in Residual % of parent Mutations activity% enzyme Pectate lyase of SEQIDNO: 45 100 2 (parent enzyme) D68* +N69* + L70* + K71* 46 102 N50L + K54V 46 102 I390N 46 102 V141N 47 104L75P 47 104 K115N 47 104 L268Y + F251I 48 107 A91E 48 107 T105P 48 107L106Q 49 109 K87A 49 109 S30T + K99I 49 109 K139I + K213N 49 109 R272Y50 111 M237I 51 113 C199N 51 113 F215Y 52 116 K213T 52 116 A228I + F251I53 118 K115I + Q146H 57 127 K257N 59 131 K213N + T258I 60 133 M122Q 61136 K386P 61 136 K218P 61 136 A332P 62 138 F251I 64 142 A305P 64 142D48P 64 142 S134L + K257E 66 147 M64F + K213T + K218L + A305P 73 162S331P 75 167 M64F + M122K + K118E + K213T + 78 173 K218L + A305P M64F +K139I + Q146H + S337C 91 202 M64F + V123I + K139I + Q146H + S337C 91 202K139I + Q146H + S337C 100 222

Example 2: Stability of Pectate Lyases in Liquid Detergent AfterProlonged Incubation

The detergent stability of the pectate lyase variants of the presentinvention is assessed by measuring the activity of the variants afterincubation of an enzyme-detergent mixture, which is described below.

Residual Activity Assay

30 micro litre of an enzyme solution (culture supernatant or purifiedenzyme) is mixed with 1 ml of typical European heavy duty liquiddetergent in two sample tubes. One of the tubes is stored on ice whilethe other is incubated at 40 degrees Celsius for 18 or 24 hours. Asreference 30 microlitre water is mixed with 1 ml of detergent andincubated on ice. After incubation, 9 ml of ice-cold water is added tothe samples, which are mixed vigorously and stored on ice until furtheranalysis.

The enzymatic activity is measured by first mixing 50 micro litre ofenzyme-detergent mixture with 5 ml of assay buffer (100 mM Tris-HCL,0.68 mM CaCl₂, pH 8.0), secondly from which solution, 75 micro litre ismixed with 75 micro litre freshly prepared substrate solution (1%polygalactoronic acid in assay buffer) and incubated at 40 degreesCelsius for 10 minutes. Thirdly, 100 micro litre of the incubationmixture is added into 100 micro litre stop-buffer (50 mM H₃PO₄) in aUV-transparent microtiterplate and the absorbance at 235 nm is measuredin a spectofotometer. The water-detergent sample is used to zero thespectofotometer.

Now the residual activity is calculated as the activity (A235absorbance) in the sample incubated at 40 degrees Celsius for 90 minutesrelative to the activity in the sample stored on ice:Residual activity (RA)=Absorbance[sample incubated at 40 degreesCelsius]/Absorbance[sample incubated at 0 degrees Celsius]×100

Thus the residual activity is equivalent to the detergent stability ofthe enzyme. Table 4 lists the improved residual activity of a number ofsubstitutions of the pectate lyase of SEQ ID NO:2 incubated in a typicalEuropean heavy duty liquid detergent for 18 hours. Likewise Table 5lists the residual activity of pectate lyase variants incubated for 24hours. TABLE 4 Substitutions in the pectate lyase of SEQ ID NO: 2 givingrise to increased residual activity, which is measured after incubationin European heavy duty liquid detergent for 18 hours. Residual Mutationsactivity % Pectate lyase of SEQIDNO: 2 (parent enzyme) <5% K139I +Q146H + S337C 22 D48P + M64F + K139I + Q146H + K213T + K218L + A305P +S337C 53 D48P + M64F + T105P + M122K + K118E + K213T + K218P + A305P +S331P 41 M64F + M122K + K118E + Q146H + K213T + K218L + A305P + S340P 38D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I + A305P +S331P 37 D48P + M64F + K99I + K118E + M122K + Q146H + K213N + K218P +A305P + S331P 24 D48P + T105P + K139N + K213T + K218P + T258I + A305P +S331P 23 D48P + M64F + K139I + Q146H + K213T + K218L + A305P + S337K 56D48P + M64F + K139I + Q146H + K213T + K218L + A305P + S337R 46 M64F +M122K + K118E + Q146H + K213T + K218L + A305P + S337K 49 M64F + M122K +K118E + Q146H + K213T + K218L + A305P + S337R 59 D48P + T105P + K139N +K213T + K218P + T258I + A305P + S331P + S337K 60 D48P + T105P + K139N +K213T + K218P + T258I + A305P + S331P + S337R 57 D48P + M64F + T105P +K139N + Q146H + K213T + K218P + T258I + A305P + S331P 32 D48P + K99I +T105P + K139N + K213T + K218P + T258I + A305P + S331P 44 D48P + M64F +L106Q + K139I + Q146H + K213T + K218L + Y234H + A305P + S331P + S337C 52D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I + A305P +S331P + S337R 80 D48P + M64F + T105P + K139I + Q146H + K213T + K218P +T258I + A305P + S331P + S340P 64 D48P + M64F + T105P + K139N + Q146H +K213T + K218P + T258I + A305P + S331P 30 D48P + M64F + T105P + K139I +Q146H + N189D + K213T + K218P + T258I + A305P + S331P + S337R 80 D48P +M64F + T105P + K139I + Q146H + K213T + K218P + T258I + A305P + S331P 32

TABLE 5 Substitutions in the pectate lyase of SEQ ID NO: 2 giving riseto increased residual activity, which is measured after incubation inEuropean heavy duty liquid detergent for 24 hours. Residual MutationsActivity % Pectate lyase of SEQIDNO: 2 (parent enzyme) <5 D48P + M64F +T105P + K139I + Q146H + K213T + K218P + T258I + A305P + S331P + S337R 67D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I + A305P +S331P + S337K + S340P 85 D48P + M64F + T105P + K139I + Q146H + N189D +K213T + K218P + T258I + S298N + A305P + S331P + S337R 77 D48P + M64F +T105P + K139I + Q146H + K148E + K213T + K218P + T258I + A305P + S331P +S337R 77 D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I +A305P + S331P + K334E + S340P 60 D48P + M64F + T105P + K139I + Q146H +K213T + K218P + T258I + A305P + S331P + K334E + S337K + S340P 90 D48P +M64F + T105P + K139N + K213T + K218P + T258I + A305P + S331P + S337K 69D48P + M64F + T105P + K139N + K213T + K218P + T258I + A305P + S331P +S337R 63 D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I +A305P + S331P + S337K 80 D48P + M64F + T105P + K139I + Q146H + K148Q +K213T + K218P + T258I + A305P + S331P + S337K 83 D48P + M64F + T105P +K139I + V141E + Q146H + K213T + K218P + T258I + A305P + S331P + S337K 88D48P + M64F + T105P + K139I + Q146H + C199S + K213T + K218P + T258I +A305P + S331P + S337K 82 D48P + M64F + T105P + K139I + Q146H + K213T +K218P + T258I + Y295H + A305P + S331P + S337K 84 D48P + M64F + T105P +K139I + Q146H + K213T + K218P + T258I + A305P + S331P + K334E + S337K 86D48P + M64F + K99R + T105P + K139I + Q146H + K213T + K218P + T258I +A305P + S331P + S337K 82

Example 3 DSC on Pectate Lyase Wild-Types and Variants

The thermal stability of pectate lyase variants of the invention wasmeasured with differential scanning calorimetry (DSC). The experimentswere conducted in a buffer (as opposed to detergent). Purified enzymesamples were dialysed twice against 5 L 20 mM HEPES buffer, 0.68 mMCaCl₂, pH 8.0 in 10 kDa cut-off Slide-A-Lyzers (Pierce, USA) andconcentrated to approx. 10 mg/ml by 2,400 g centrifugation in a BiofugeStratos (Kendro, Germany) in YM 10 kDa Centricon cells (Amicon). DSC wasrun in a MCS 4100 (Calorimetry Sciences Corporation, USA) with a 1°C./min gradient from 5 to 110° C. and data analysed by CpCalc 2.1software (Applied Thermodynamics, USA). TABLE 6 Transition temperatures(T_(t)) for the tested pectate lyase enzymes. Mutations T_(t) (° C.)Parent 67.6 D48P + M64F + T105P + K139I + Q146H + K213T + K218P +T258I + A305P + S331P 71.5 K139I + Q146H + S337C 72.4 D48P + M64F +T105P + K139I + Q146H + K213T + K218P + T258I + A305P + S331P + S340P74.1 D48P + M64F + T105P + K139I + Q146H + K213T + K218P + T258I +A305P + S331P + K334E + S337K + S340P 74.2 M64F + K139I + Q146H + S337C74.5 D48P + M64F + T105P + K139I + Q146H + N189D + K213T + K218P +T258I + S298N + A305P + S331P + S337R 74.7 D48P + M64F + T105P + K139I +Q146H + K213T + K218P + T258I + A305P + S331P + S337K 75.0 D48P + M64F +T105P + K139I + Q146H + K213T + K218P + T258I + A305P + S331P + S337R75.6 D48P + M64F + T105P + K139I + Q146H + K148E + K213T + K218P +T258I + A305P + S331P + S337R 76.6 D48P + M64F + T105P + K139I + Q146H +K213T + K218P + T258I + A305P + S331P + S337K + S340P 77.7

It is noted that that all variants are thermally stabilised compared tothe parent.

1-30. (canceled)
 31. A variant of a parent enzyme having pectate lyaseactivity (EC 4.2.2.2), which variant has improved detergent stabilitycompared to said parent enzyme, and which variant comprises analteration at one or more positions selected from the group consistingof positions number: 5, 9, 11, 26, 28, 30, 31, 37, 40, 45, 46, 47, 48,49, 50, 51, 52, 54, 61, 64, 68, 69, 70, 71, 74, 75, 76, 79, 86, 87, 91,99, 105, 106, 107, 111, 115, 116, 118, 122, 123, 134, 136, 139, 140,141, 146, 148, 156, 158, 170, 182, 185, 186, 189, 193, 194, 196, 199,201, 202, 204, 213, 215, 218, 224, 228, 229, 234, 235, 237, 251, 256,257, 258, 272, 277, 286, 295, 298, 301, 302, 303, 305, 307, 308, 314,316, 323, 324, 326, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340,341, 349, 356, 357, 363, 366, 378, 381, 384, 386, 387, 389, 390, 391,393 and 397, wherein the alteration(s) are independently (i) aninsertion of an amino acid downstream of the amino acid which occupiesthe position, (ii) a deletion of the amino acid which occupies theposition, or (iii) a substitution of the amino acid which occupies theposition with a different amino acid, and wherein each positioncorresponds to a position of the amino acid sequence of the pectatelyase having the amino acid sequence of SEQ ID NO: 2, and wherein theparent enzyme is the pectate lyase shown on SEQ ID NO: 2 or a pectatelyase having at least 65% identity to the amino acid sequence of SEQ IDNO:
 2. 32. A variant of claim 31, wherein the alteration(s) aresubstitution(s).
 33. A variant of claim 31, wherein the alternation(s)are one or more substitutions selected from the group consisting of:H5R, E9G, N11Y, K26Q, S28T, S30F, S30P, S30T, H31N, N37D, Q40E, L45V,G46D, K47N, K47R, D48E, D48P, T49P, N50D, N50L, N50Y, N51Y, T52M, K54V,T61A, M64F, D68*, N69*, L70*, K71*, K71E, G74D, L75A, L75P, N76D, K79A,D86N, K87A, K87E, A91E, K99I, K99N, K99R, T105A, T105P, L106Q, E107K,A111E, K115A, K115I, K115N, K115Q, N116D, K118A, K118E, M122E, M122K,M122N, M122Q, V123I, S134L, T136S, K139E, K139F, K139I, K139M, K139N,K139S, I140V, V141G, V141E, V141L, V141N, Q146F, Q146H, Q146I, Q146V,K148E, K148Q, N156S, E158N, D170N, Q182D, Q182E, N185H, N186H, N189D,H193Y, I194V, I196V, C199N, C199S, F201L, N202K, G204R, K213E, K213N,K213T, F215Y, K218E, K218L, K218P, G224S, A228I, S229T, Y234H, I235V,M237I, F251I, S256C, K257E, K257N, T258I, L286Y, R272C, R272H, R272Y,V277D, G286A, Y295H, S298N, S301Y, S302A, D303S, A305P, S307R, Y308S,K314N, S316F, N323M, V324A, D326N, S331P, S331T, A332P, A333E, K334E,T335S, T335R, I336S, S337C, S337K, S337L, S337R, V338E, V338Y, F339I,S340A, S340K, S340N, S340P, S340Q, G341S, G349R, Q356H, I357V, N363S,S366N, T378G, T378S, A381D, H384N, K386P, K386R, S387A, V389I, I390N,I390T, S391N, A393V and K397D.
 34. A variant of claim 31, wherein theparent pectate lyase has an amino acid sequence which has a degree ofidentity to the amino acid sequence of SEQ ID NO: 2 of at least about70%.
 35. A variant of claim 31, wherein the parent pectate lyase isencoded by a polynucleotide which hybridizes under medium stringencyconditions with the polynucleotide of SEQ ID NO: 1 or its complementarystrand.
 36. A variant of claim 31, wherein the parent pectate lyase is awild-type pectate lyase.
 37. A variant of claim 31, wherein the parentpectate lyase is a Bacillus pectate lyase.
 38. A variant of claim 37,wherein the parent pectate lyase is encoded by the polynucleotidepresent in the plasmid of the strain Bacillus subtilis DSM
 14218. 39. Avariant of claim 31, wherein the total number of alterations isthirteen.
 40. A cleaning or detergent composition, comprising a variantof claim 31 and a surfactant.
 41. A composition of claim 40, whichadditionally comprises a cellulase, a lipase, a cutinase, anoxidoreductase, a protease, an amylase, a hemicellulase, such as amannanase, a xylanase, a galactanase, an arabinofuranosidase, anesterase, a lichenase, an arabinanases, another pectate lyase or amixture thereof.
 42. An enzymatic scouring method, comprising contactingcell-wall material with a variant of claim
 31. 43. A method forenzymatic removal of cell-wall material from textile, comprisingcontacting the textile with a variant of claim
 31. 44. A variant of aparent enzyme having pectate lyase activity (EC 4.2.2.2), which varianthas improved detergent stability compared to said parent enzyme, andwherein (a) the overall charge of the enzyme has been made morenegative; (b) oxidation labile amino acid residues have been replaced;(c) more flexible amino acid residues have been replaced with lessflexible amino acid residues; and/or (d) the variant has improvedcalcium-depletion stability.
 45. A polynucleotide encoding a variant ofclaim
 31. 46. An expression vector comprising the polynucleotide ofclaim
 45. 47. A microbial host cell transformed with the expressionvector of claim
 46. 48. A microbial host cell of claim 47, wherein thecell is a bacterium.
 49. A microbial host cell of claim 47, wherein thecell is a fungus or yeast.
 50. A method for producing a variant,comprising (a) culturing a microbial host cell of claim 47 underconditions conductive to the expression and secretion of the variant,and (b) recovering the variant.